Crown-gall Tumor Initiation Research Articles

The Receiver of the Agrobacterium tumefaciens VirA Histidine Kinase Forms a Stable Interaction with VirG to Activate Virulence Gene Expression.

The plant pathogen Agrobacterium tumefaciens carries a virulence gene system that is required for the initiation of crown gall tumors on susceptible plants. Expression of the vir genes is activated by the VirA/VirG two component regulatory system. VirA is a histidine kinase which signals the presence of certain chemicals found at the site of a plant wound. The receiver domain located at its carboxyl terminus defines VirA as a hybrid histidine kinase. Here, we show that the VirA receiver interacts with the DNA-binding domain of VirG. This finding supports the hypothesis that the receiver acts as a recruiting factor for VirG. In addition, we show that removal of the VirA receiver allowed vir gene expression in response to glucose in a dose dependent manner, indicating that the receiver controls VirG activation and suggesting that the supplementary ChvE-sugar signal increases this activity.

Antitumor properties of two traditional aromatic rice genotypes (Kalijira and Chinigura).

Methanol extract of bran and unpolished grain of two traditional aromatic rice genotypes viz. Kalijira and Chinigura were assayed for their activity on the growth and initiation of crown-gall tumors on potato disks. Three Agrobacterium tumefaciens (A. tumefaciens) strain AtSl0105, AtTa0112, and AtAc0114 were used as the tumor forming agent. Collected rice was separated to bran and unpolished grain by different milling processes and made into fine powder before extracting using methanol. Antitumor assay of plant extracts was performed according to standard potato disc bioassay. Disc diffusion assay (Kirby-Bauer Method) was used to screen A. tumefaciens sensitivity test. The results demonstrated a high correlation between the ability of aromatic rice to inhibit the initiation and growth of crown-gall tumors on potato disks. Maximum tumor inhibitions were observed against the strain AtSl0105 by Kalijira bran (73.91%) and Chinigura bran (69.56%). Both unpolished grains showed significant effect (Kalijira 57.43%, Chinigura 55.53%) to inhibit the tumor. It can be concluded that aromatic rice (Kalijira and Chinigura) might be a potential source of antitumor agent that can be used for further drug development for tumor treatment.

Activity of the Agrobacterium T-DNA transfer machinery is affected by virB gene products.

The oriT (origin of transfer) sequence and mob (mobilization) genes of plasmid RSF1010 can functionally replace transfer DNA (T-DNA) borders to generate an RSF1010 intermediate transferable to plants through activities of the tumor-inducing (Ti)-plasmid virulence (vir) genes of Agrobacterium tumefaciens. Because the Ti plasmid virB gene products are hypothesized to form a membrane-localized T-DNA transport apparatus, we investigated whether specific virB genes were needed for RSF1010 transfer. Here we report that transformation of Nicotiana tabacum leaf explants by an RSF1010-derivative plasmid (pJW323) requires the essential virulence genes virB9, virB10, and virB11, consistent with the hypothesis that both the T-DNA and RSF1010 transfer intermediates utilize the same transport machinery. Further, while pJW323 is transferred into plant cells by Agrobacterium strains harboring both pJW323 and pTiA6, the initiation of crown gall tumors (i.e., T-DNA transfer) is greatly suppressed. Coordinate overexpression of the virB9, virB10, and virB11 gene products relieves pJW323-mediated oncogenic suppression and restores tumorigenicity, but does not increase the transfer frequency of pJW323 into plant cells. We propose that the virB9, virB10, and virB11 gene products function coordinately and stoichiometrically to enhance DNA transfer in a fashion specific for the T-DNA intermediate.

Influence of a crown-gall tumor initiation enhancer on bacterial attachment to the host plant cell wall

The competitive activities of different plant cell walls upon Agrobacterium tumefaciens attachment have been studied in vitro by means of two crown-gall tumor initiation assays. The low or high susceptibility of different plant species is independent of their capacity to cause bacterial cells to adhere to specific sites on the plant cell walls. However, the attachment properties of cell wall fragments derived from Helianthus cotyledons seem to be age-dependent. It is found that a tumor initiation enhancer, present in extract fractions derived from highly susceptible plants and closely related with the competence for tumor formation, does not influence bacterial adherence. The two steps, attachment and the step by which the tumor initiation enhancer is involved, clearly differ in the processes leading to the transformation of a normal cell into a tumor cell.

Evidence for the existence of a universal crown-gall tumor initiation enhancer

Tumor initiation of different dicotyledonous plant species inoculated with Agrobacterium tumefaciens B6 has been studied in vivo and in vitro. The tumor formation in weakly susceptible plants can be strongly enhanced by exogenously applied active extract fractions derived from highly susceptible Helianthus cotyledons. It is found that highly susceptible plants (Kalanchoë, Lycopersicon and Pinto beans) contain an active tumor initiation enhancer which is clearly similar to the compound(s) found in Helianthus cotyledons. No activity can be detected in extracts derived from weakly susceptible plants (Coleus, Phaseolus) or in those obtained from crown-gall tumor tissues.

Enhancement of Crown-Gall Tumor Initiation by Extract Fractions from Tumor Forming Cotyledons of Helianthus giganteus L

Summary Tumor initiation in cotyledons of Helianthus giganteus L. inoculated with Agrobacterium tumefaciens B 6 has been studied in vitro . The susceptibility of the cotyledons can be enhanced by exogenously applied active extract fractions derived from 6-days-old cotyledons. About the same results could be obtained with infections on the epi- and hypocotyl of Helianthus plants in vivo . The growth of the tumors was unaffected by these treatments. It is found that the susceptibility of the cotyledons for tumor initiation is clearly correlated with the endogenous content of the active compound(s). This active compound(s), present in the extract fractions, does not require the immediate presence of the bacteria to be physiologically active. No influence of the active compound(s) on the virulence of the bacteria has been observed.

The Crown-Gall Potato Disk Bioassay as a Primary Screen for Compounds With Antitumor Activity

A variety of samples consisting of purified compounds (of various origins) and ethanol extracts from plants were assayed for their activity on the growth and initiation of crown-gall tumors on potato disks. The results demonstrated a high correlation between the ability of these compounds to inhibit the initiation and growth of crown-gall tumors on potato disks and their corresponding activity on the mouse P388 leukemia protocol. Of the 21 compounds tested to date, there were two false-positives and one false-negative.

Cell surface carbohydrates of [formula omitted] involved in adherence during crown gall tumor initiation

N-acetyl-D-galactosamine and β-D-galactose, located in the O-antigenic region of Agrobacterium lipopolysaccharide, are involved in host-bacterium adherence during crown gall tumor initiation as indicated by fluorescence spectroscopy studies.

Crown Gall Tumor Disc Bioassay

Seventeen samples consisting of purified compounds and various ethanol extracts from plant sources were tested for activity on the initiation of crown gall tumors on potato discs. The results demonstrated definite correlation between the ability of these samples to inhibit the formation of crown gall tumors and their activity on the P388 leukemia system in mice. Samples showing only cytotoxic effects in KB cell cultures did not affect tumor initiation in our system. The active materials had no effects on bacterial viability or on the ability of the bacteria to attach to a tumorbinding site.

Cell Walls of Crown-Gall Tumors and Embryonic Plant Tissues Lack Agrobacterium Adherence Sites

Crown-gall tumor initiation by Agrobacterium tumefaciens is inhibited by cell walls from normal dicotyledonous plants but not by cell walls from crown-gall tumors apparently because of bacterial adherence or nonadherence, respectively, to the different cell walls. Cell walls from normal and tumor tissues in culture also show this difference, indicating that the two types of tissue stably maintain this difference under these conditions. Habituated tissue cultures, which resemble crown-gall tumor cultures, however, form cell walls that are inhibitory like those of the normal cultures from which they are derived. Monocotyledonous plants do not act as hosts for Agrobacterium and bacteria-specific inhibition is not shown by cell walls from several species of grass, a monocot family. Cell wallsfrom "embryonic" tissues (dicot seedlings less than 2 centimeters long), unlike those from older seedlings, are non-inhibitory. Crown-gall tumors thus resemble embryonic tissues in this respect.

On the question of crown-gall tumor initiation by DNA of bacteriophage PS8.

DNA of Agrobacterium tumefaciens bacteriophage PS8 was isolated by using several procedures. Whole phage and phage DNA were tested for tumor-inducing ability on 10 species of plants with various additions to assist such activity. The reported tumorigenicity of phage PS8 DNA could not be confirmed, and no evidence to implicate phage PS8 involvement in tumor initiation was obtained.

Bacterial attachment to a specific wound site as an essential stage in tumor initiation by Agrobacterium tumefaciens.

The number of tumors initiated by Agrobacterium tumefaciens strain B6 on primary pinto bean leaves was decreased when cells of an avirulent strain (IIBNV6) were included in the inoculum. With sufficient B6 cells to initiate ca. 50% of the maximal number of tumors per leaf, inhibition was detected at a 1:1 ratio of B6 to IIBNV6 cells and increased linearly with the logarithm of the number of IIBNV6. Varying the number of B6 in the presence of a constant number of IIBNV6 or varying the number of both, while maintaining a constant ratio of B6 to IIBNV6, showed that the inhibition was a function of the absolute concentration of each cell type. The data fit a one-particle dose response curve, which indicates that a single IIBNV6 cell can prevent tumor initiation by a single B6 cell. Inhibition was obtained with mixed inocula and when the addition of IIBNV6 preceded B6, but not when B6 preceded IIBNV6. Heat-inactivated IIBNV6 inhibited, as did ultraviolet or heat-inactivated B6. Several unrelated bacteria and certain strains of Agrobacterium failed to inhibit, whereas other related strains gave inhibition. Attachment of IIBNV6 to a specific would site, thus excluding B6 from the site, is proposed to account for these data. A specific complementary binding of a virulent bacterium to a host wound site exposed by the inoculation procedure is suggested as an essential early event in the crown-gall tumor initiation process.

Promotion of crown-gall tumor initiation on primary pinto bean leaves by certain inorganic salts

Promotion of crown-gall tumor initiation on primary pinto bean leaves by certain inorganic salts