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Crown Gall Disease and Agrobacterium Tumefaciens: A Study of the History, Present Knowledge, Missing Information, and Impact on Molecular Genetics.

I. Abstract

The production of crown gall tumors in plants caused by Agrobacterium tumefaciens represents a unique disease involving the transfer of DNA from the bacterium to the nucleus of the plant. Vital aspects of this transfer are still being studied.

II. Introduction

A bacteria-caused disease in grapevines was first observed in the 1850s, but the relationship to a known bacterium was not understood until the early 1900s (Smith & Townsend, 1907). The diseased plants produced external galls or tumorous growths along the crown (base) of the stem of dicotyledonous plants such as tobacco (Butcher, 1977). The specific Agrobacterium bacterial genus and species involved were finally identified as the phytopathogen Agrobacterium tumefaciens. This Agrobacterium genus of about 4 species is found in the Rhizobiaceae family, along with the Rhizobium genus of nitrogen fixers (Fig. 1) (Powell & Gordon, 1989). Research into this disease, considered related to but different from animal cancers, continues today. So far, it is a unique disease involving the transfer of DNA from a prokaryote into chromosomes of plant eukaryotes by mechanisms that are still incompletely known but that has permitted a new area of plant-genetic engineering leading to the production of numerous transgenic plant s as a result of gene transfer from one plant to another. This uniqueness and the methods permitting genetic transfer of foreign genes to many undiseased dicotyledonous and even to some monocotyledonous species provide an unusual opportunity for a case study of one example of modern plant research that involves many disciplines (Tinland, 1996). Crown gall tumors vary considerably in their morphology, ranging from recognizable stem or root tissue to a chaotic mixture of cell types (Drummond, 1979). Crown gall disease is one of the four best-analyzed neoplastic diseases of plants. Other forms are Black's wound tumor disease, caused by an RNA virus; Kostoff genetic tumors, caused by chromosomal imbalance; and habituation (Braun, 1982). Neoplastic galls are also produced by three phytopathogenic bacteria in addition to A. tumefaciens, but these require either the continued presence of the bacteria or a supply of auxin and cytokinin (Morris, 1995).

Crown gall tumors can be maintained as undifferentiated masses of tumor cells in tissue cultures without the infecting bacteria and added hormones required for cell culture of normal tissues. Upon grafting onto an intact shoot, some develop meristematic zones of cells that initiate the formation of small, distorted shoots, or teratomas (Lippincott & Lippincott, 1981; Turgeon, 1982). In other cases, the shoots formed are similar to those of normal plants but retain the capacity for tumor growth. They can even appear to have completely recovered from the transformed state. Teratoma structures of shoots, however, have a short life span; the stems die, and the tumors decay. Teratomas of intact shoots are separated from the vascular system by the tumor mass. The results of grafting of teratoma shoots into the cambium of healthy host plants is variable, but shoots can become more regular in appearance, with an orderly developmental sequence. Normalization of structure upon grafting depends on the establishment of an effective vascular connection. They can even ultimately flower and set viable seed when meiosis occurs, so that completely normal plants can be formed.

Most studies now are with cell cultures and grafted plants, not with natural crown galls found mainly at the crown base. The present uniqueness of DNA transfer from a prokaryote to a eukaryote certainly highlights these growths.

III. Major Stages in Crown Gall Disease

Major stages in the infection by Agrobacterium tumefaciens and the subsequent formation of tumorous galls on the stems of plants involve a series of responses between an individual bacterium (A. tumefaciens) and mainly dicotyledonous plants (Binns & Thomashow, 1988). Wounding by insects, other animal bites, or human mechanical piercing of the surface of a stem induces limited cell division to form a callus layer as a common defense mechanism, involving the secretion of phenolic defense compounds. These divisions will cease within a few days unless the appropriate bacteria are present. A chemotactic response of A. tumefaciens on the surface of a callus cell toward specific phenolics and carbohydrates in the callus exudate involves a signal to the constitutive protein of the virA gene in the Ti-plasmid that phosphorylates a second constitutive protein, followed by the activation of other vir genes to produce a sequence of proteins. T-DNA processing involves the excision from the Ti-plasmid of a T-strand, consi sting of single DNA molecules, that is transported into the plant nuclear genome (Tinland, 1996). The T-DNA oncogenes produce uncontrolled proliferation of crown gall cells via the production of auxins and cytokinins for the dividing plant cells and specific opines that are secreted as an energy source for the attached bacterium, but mainly for the surrounding aggregation of A. tumefaciens attracted by the appropriate opine type.

IV. Historical Record of Crown Gall Disease

A list of the approximate dates of key discoveries in the historical record of crown gall disease since about 1900 is shown in Table I. Useful reviews exist for earlier discoveries up to about 1980 (Braun, 1978; Davey et al., 1994; de Ropp, 1951; Drummond, 1979; Lippincott & Lippincott, 1981) and for the more recent history (Binns & Thomashow, 1988; Burdock & Hooykaas, 1998; Hooykaas & Schilperoot, 1992; Lindsay & Jones, 1990; Powell & Gordon, 1989; Tinland, 1996; Zupan & Zambryski, 1997). Braun's history of the crown gall disease (Braun, 1982) is of particular interest because he was a vital experimentalist with many significant publications from about 1940 through the 1970s. He established the use of tissue-culture techniques in the study of crown gall disease. The history can be divided into two major phases: a basic biological phase that includes the taxonomy, physiology, and genetics of both the bacterium and the contaminated plants, followed by a molecular genetic phase that involves both the transfer of T-DNA from bacterial plasmids to the plant nucleus and the subsequent transfer of DNA, leading to the biosynthesis of hormones and opines in the plant's cytoplasm, followed by the secretion of the opines to return the cycle to the surrounding bacteria. However, major aspects of the identification of the components involved are still unknown.

The disease that produces these galls was described in grapevines in 1882 and was subsequently studied in a variety of natural plants (Powell & Gordon, 1989). From about 1940 on, studies were based on tumors in cell cultures and grafts on plants (Braun, 1982). Agrobacterium tumefaciens (originally Bacterium phytomonas, and then Phytomonas tumefaciens) was identified as the agent of transmission in the 1930s (de Ropp, 1951; Link et al., 1937). Smith and Townsend (1907) were among the earlier major investigators of crown gall disease (Braun, 1954, 1982). A plant hormone connection with the disease was based on the fact that exogenous auxins and cell-division factors were not required for cell cultures of tumors but were for nontumorous cultures (Brown & Gardner, 1936; Klein & Link, 1952). The hormones accumulating in infected plant cells were identified as [beta]-indoleacetic acid (IAA) in 1937, and a cell-division factor were later identified as a cytokinin (Link et al., 1937). Secondary tumors were observed well above the crown of the stem, with no apparent connection to the primary tumors (de Ropp, 1951; Lippincott & Lippincott, 1981). Tumors grafted to normal plants were found free of crown gall bacteria (Braun, 1954; Butcher, 1977; Zerbak, 1989). By the 1940s, the disease was referred to sometimes as a cancer but more often as an oncogenic transformation of normal cells into autonomous tumors (de Ropp, 1951). The term TIP as the unknown tumor-inducing principle transmitted upon bacterial infection was introduced in the late 1940s (Braun, 1954, 1982).

By the 1950s, arguments concerning the nature of TIP arose. DNA was suspected, but the data were inadequate (Braun, 1982). DNA, however, was known to be present in plasmids. The retention of the autonomous state on hormone-free culture media was thought to be evidence that TIP was being transmitted to the plant cells, but better evidence did not appear until the 1970s. Tumorous growth could sometimes be reversed by grafting tumor culture (teratoma) onto a healthy plant, the seed of which formed normal plants apparently upon meiosis (Braun, 1954, 1965; Butcher, 1977). A [N.sup.6]-substituted adenine derivative (6-furfurylaminopurine) derived from the breakdown of sperm DNA was considered a possible active cell-division factor (called kinetin), but there was considerable disagreement as to the nature of the replacement for the original coconut-milk mixture used in earlier studies along with an auxin (Braun, 1982). A variety of natural cell-division factors, now called cytokinins, as N-substituted adenine-conta ining compounds in plants were finally identified in the 1960s; zeatin was the first (Zambryski, 1992). Genes that encode the biosynthetic enzymes for either the plant or bacterial pathway of either auxins or cytokinins were still unknown (Morris, 1986, 1995). One of the first unusual amino acids called opines, lysopine, was identified in A. tumefaciens in 1956 (Braun, 1982).

Adherence of A. tumefaciens to a site in the wound was shown as the earliest detectable stage in tumor formation in the 1960s (Lippincott & Lippincott, 1981). Nucleic acid hybridization studies to determine the genetic involvement in crown gall tumors and the nature of TIP were made, but no concrete evidence of bacterial DNA sequences in sterile crown gall tissue was found (Butcher, 1977). A variety of opines, unusual amino acids, such as octopine and nopaline, found in tumor tissue cultures but not in normal, habituated, or genetic tumor cultures, were identified in the late 1960s and early 1970s.

Whereas the initial phase of knowledge about crown gall tumors was concerned with the general external description and cell culture without exogenous hormones, the second major phase, in the 1970s and into the 1980s, described a unique process in which a DNA component of a bacterial plasmid was transferred into the nucleus of an eukaryote organism to produce the hormones for plant-cell growth and opines for bacterial consumption. Plasmid studies in the 1970s identified a series of different types of opines associated with specific plasmids and areas of the T-DNA. Virulent strains of A. tumefaciens were classified according to their genetic ability to form different opines. Such virulent strains contain large plasmids and, therefore, high extrachromosomal DNA (Butcher, 1977). By 1974 the so-called TDNA was identified in plasmids as the source of TIP (Braun, 1982; Nester et al., 1984; Powell & Gordon, 1989). Various stages of the infection process became clear (Lippincott & Lippincott, 1981): wounding was foll owed by a chemotactic response resulting in adherence of the bacterium to the plant cell; tumors were detected 2--6 days after this initial adherence, and the bacterium was subsequently not required for the tumor development. Carbohydrates of the plant cell wall were postulated to be involved in the adherence of the bacterium to the wall (Braun, 1954; Lippincott & Lippincott, 1981). The related Ri-plasmid involved in root proliferation by A. rhizogenes (hairy root disease) was identified (Davey et al., 1994; Powell & Gordon, 1989).

Experimental evidence of the essential role of T-DNA in the transformation process in crown gall disease was published in 1980 by several scientific groups (Braun, 1982; Yadav et al., 1982). The 1980s might be considered the beginning of the era of molecular genetics involving crown gall disease (Figs. 2, 3) (Binns & Thomashow, 1988; Christou, 1996; Davey et al., 1994; Gasser & Fraley, 1992; Herrara-Estrella et al., 1983; Hooykaas & Schilperoot, 1984, 1992; Powell & Gordon, 1989). A. tumefaciens plasmid genes in the T-DNA that code for a series of three oncogenic genes (sometimes called onc genes), involving the biosynthesis of hormones such as auxin (IAA) (tms1 & tms2A or iaaM & iaaH)in A. tumefaciens and cytokinin (ipt gene), were identified (Morris, 1986, 1995). Comparable auxin genes (aux1 & 2) were identified in plasmids of A. rhizogenes (Gaudin & Fraley, 1993). Proteins produced by vir genes involved in a complex set of reactions were identified in 1983--1986. Major regions within the infective plasmid s were identified: the vir region, the T-DNA region (transferred to the plant genome), and catabolic regions (not transferred) (Powell & Gordon, 1989). The constitutive VirA protein induced by plant carbohydrate and phenolic signals in plant exudates to form a series of Vir proteins was shown to be sensed directly by the phenolic, such as acetosyringone released upon wounding (Lee at al., 1995; Palmer & Shaw, 1992). A bacterial type of conjugation was suspected as the means of the T-DNA transfer to plant cells. A protein-lipopolysaccharide on the outer membrane of the bacterium binds to a protein-pectin receptor site on the plant cell (Davey et al., 1994).

Genetic engineering involving transfer of foreign genes into plants by means of the addition of the new genes via T-DNA of Ti-plasmids as vectors (recombinant DNA techniques) was first developed from about 1985 to 1987 and is now a major technique to produce transgenic crops with new traits and to study the effects of the introduction of foreign DNA portions into another species (Hooykaas & Schilperoot, 1984, 1992). The study of transgenic plants and genetic engineering continues, and its history is still unfinished (Christou, 1996; Davey et al., 1994; Gasser & Fraley, 1992).

In the 1990s, T-DNA restriction maps were constructed (Davey et al., 1994). Functions of the nonplasmid chromosomal gene, chv, and those of the plasmid DNA were described (Sheng & Citovsky, 1996). Major advances were made in the area of the vir genes and their proteins. The protein of the constitutive virA gene is involved in phenolic sensing; the type of phenolic varies with the bacterial strain (Lee et al., 1995; Palmer & Shaw, 1992). The VirA protein was shown to function as a protein kinase and phosphotransferase. The virA protein responds chemotactically to certain types of phenolics, such as the C6-C3 phenolic compound, acetosyringone (Davey et al., 1994). Some of the functions of Vir B, D, and E proteins of the nonconstitutive genes activated by the constitutive VirA to G protein regulatory system were elucidated. VirD2 protein becomes attached to the 5' end of the T-strand; VirC coats the single-stranded T-DNA as a protectant against nucleases. The T-strand is guided through the nuclear pore of the p lant nucleus by VirB protein (Tinland, 1996). Opines were further classified according to the bacterial host loci and their catabolic and synthetic loci in the DNA, and chemotactic characteristics were studied (Kim & Farrand, 1996, 1998; Kim et al., 1996). Conjugal areas of plasmids in the Rhizobiaceae are compared in Figure 4, with detail of the gene loci involved, the tra and trb operons (Farrand, 1998). The rol gene promoters in A. rhizogenes were also analyzed (Nilsson & Olsson, 1997; Nilsson et al., 1993, 1997). Major developments in transformation technology of crops were made, involving basic enzymology and genetics.

Research on the main details of crown gall disease may be completed in the 2000s, but major information is still needed regarding the incorporation of the T-strand into the plant nucleus and the subsequent steps producing plant hormones and opines in the plant cytoplasm. The contribution of crown gall studies to our basic knowledge of biology via transgenic studies and plant engineering will continue.

V. Present Understanding of Crown Gall Disease

Much is now known about crown gall disease in many dicotyledonous plants, but many aspects of the infection process are still unknown. Basic characteristics of the overall process in which T-DNA by A. tumefaciens results in this infectious disease are summarized in Fig. 2, based on a series of references (Binns & Thomashow, 1988; Bundock & Hooykaas, 1998; Cook et al., 1997; Gelvin, 1990, 1992; Kim & Farrand, 1996, 1998; Kim et al., 1996; Tinland, 1996).

Wounding by insects, other animal bites, or human mechanical piercing of the surface of a stem induces cell division to form a thin callus layer. This callus formation is a common defense mechanism, involving the secretion of phenolics as defense compounds. The cell divisions producing the callus will cease within a few days unless the appropriate virulent strains of Agrobacterium tumefaciens bacteria are present. Phenolic compounds and carbohydrate compounds on the surface of the callus cells from this exudate act as general chemotactic signals for attachment of virulent strains of the bacteria to the plant stem (B[acute{e}]langer et al., 1997; Gelvin, 1992; Lee at al., 1995; Palmer & Shaw, 1992). The phenolics exuded, however, have not been effectively distinguished from routine defense reactions. Lignin-like components (lignans) have been cited, but they are also typical defense-induced phenolics. Subsequent VirA protein activation by a second set of more specific chemotactic signals, phenolics and carboh ydrates, will be discussed below (Dy[acute{e}] & Delmotte, 1997; Lee et al, 1995; Palmer & Shaw, 1992). Attachment of single bacteria to cells of the wound site is followed by a massive aggregation of other bacteria (colonization) at the plant-cell surface, involving conjugal transfers between bacteria upon attraction by opine secretions (Binns & Thomashow, 1988). The aggregates result after the formation of cellulose fibrils by the bacteria. A pectin associated with a glycoprotein receptor has been implicated as the plant cell-wall receptor. A lipopolysaccharide is involved as the bacterial receptor. The initial attachment, followed by a series of cellular responses of both the bacterium and plant, leads to transformation of plant cells into tumorous galls. Bacteria disappear from the wound before the occurrence of tumor formation. Indirect evidence indicates that a single attached bacterium can transform 30 or more surrounding cells, so that about 20 copies of the T-DNA are present in each of the 30 cells ( Lippincott & Lippincott, 1981). Flavonoids act as regulators of plant development: competitive root colonization via isoflavonoids released by alfalfa roots serves as a defense technique, and an A. tumefaciens strain has a genetic locus involving a pumping device that reduces the accumulation of coumestrol (Palumbo et al., 1998; Spaink, 1998).

The morphology of bacteria-induced wounds in sunflower stems injured by toothpicks contaminated with A. tumefaciens does not appear different from that of only mechanical wounding during the first 6 hours, but bacteria can be observed between dead cells as seen by electron microscopy (Chi & Smuckler, 1981). Bacteria can no longer be observed after 24 hours. Tumor nodules can be noted by 4 to 5 days. Micrographs indicate a haphazard structure in or extending from vascular bundles. A wide zone of undifferentiated cells resembling the vascular cambium can be observed extending into the adjacent parenchyma. Growth continues, with the expanding mass forcing aside the adjacent parenchyma cells and other vascular bundles. The cell pattern becomes more irregular, with cells varying in size, shape, and staining reactions.

The formation of secondary tumors above the crown level was observed in the 1930s. These secondary tumors arise at some distance from the initial wound inoculation without any apparent cellular connection with the primary gall, but some A. tumefaciens are known to spread long distances via vascular tissues (Lippincott & Lippincott, 1981; Turgeon, 1982) The presence or absence of bacteria, however, has not been resolved, and no completely satisfactory explanation for this phenomenon can yet be given.

Figure 2 gives a general scheme of the sequence of reactions in crown gall disease leading up to the formation of the nodule on the external surface of the stem. Most of the time data were determined in cultivated plants using the "prick method," followed by application of the bacterium with varied plasmid and opine types, not ones in nature, and are variable in the different plants studied. About 4 to 5 days are required to initiate the burst of cell divisions leading to a visible nodule on the surface of a young plant, involving the transfer of T-DNA from A. tumefaciens to the external surface of the shoot of a plant, commonly a dicotyledonous plant (Binns & Thomashow, 1988; Chi & Smuckler, 1981; Lippincott & Lippincott, 1981). By about 6 weeks (42 days) after the attachment of a bacterium, nodules are easily visible on the surface of the stem. They are variable in internal structure. Some remain as an undifferentiated mass of new cells with an ill-defined internal vascular structure. Other nodules contain differentiated areas in the central mass, with visible sieve plates and other vascular elements. These differences vary with the plant studied (sunflower in Chi & Smuckler, 1981; Kalanchoe and carrots in Lippincott & Lippincott, 1981). Upon wounding, a "window of competence," ranging from 24 to 120 hours, occurs prior to the burst of cell divisions that produce the nodule in about 6 weeks (Binns & Thomashow, 1988; Chi & Smuckler, 1981). Bacteria adhering to a wound cell of the stem can be observed for variable amounts of time, from 6 to 18 hours. The morphology of the stem surface is not different from that of a sterile "prick" for about the first 6 hours or even up to 24 hours. Most of the opines are secreted to the bacterial aggregations surrounding the nodule. Regulatory systems can be quite complex (Piper et al., 1999).

Three genetic regions of the bacterium are now known to contribute to the transfer and integration of this disease: the virulence (vir) region of the plasmid, the segment of the plasmid transferred from the bacterium to the plant (T-DNA), and a set of chromosomal virulence genes (chv) (Fig. 2). The sensor protein, VirA, of the bacterium is a homodimer that directly detects small phenolic compounds released by wounded cells in the callus exudate, resulting in autophosphorylation of the VirA protein of the constitutively expressed virA gene in the bacterium (B[acute{e}]langer et al., 1997; Dy[acute{e}] & Delmotte, 1997; Lee et al., 1995; Zupan & Zambryski, 1997). VirA is an inner-membrane-spanning protein with a periplasmic and a cytoplasmic domain, the latter being responsible for the recognition of a composite plant signal consisting of a phenolic compound and a sugar. VirA phosphorylates the protein expressed by the constitutive virG gene. The VirG protein then activates the remainder of the vir genes of th e plasmid to produce proteins B, D, and E (Figs. 2, 3) (Sheng et al., 1996; Zupan & Zambryski, 1997). Phosphorylation of both VirA and VirG proteins is necessary for the chemotactic response to the phenolic acetosyringone (Palmer & Shaw, 1992). The effective phenolic inducers of the VirA protein vary with the strain of A. tumefaciens, and the phenolic inducer is sensed directly by the VirA protein (Dy[acute{e}] & Delmotte, 1997; Lee et al., 1995). Three major groups of phenolics have been studied, which vary in the degree of hydroxylation and methylation of the aromatic ring (Lee et al., 1995). Acetosyringone and syringaldehyde make up one group; vanillin & ferulic acid, a second group; and p-coumaric and 4-hydroxybenzoic acid, a third group. A comparison of the known aspects of the plasmid DNA of the vir system compared with the conjugal transfer system will be summarized below.

The chv chromosomal virulence series of genes of the bacterial chromosome are mainly involved during the early stages of infection in various aspects regarding the attachment of the bacterium to the plant wall (Sheng et al., 1996; Zupan & Zambryski, 1997). ChvA and ChvB proteins are associated with the binding of the bacterium to the host-cell surface receptors (cell-cell recognition), whereas ChvE protein is a part of the signal-transduction system. The protein of the chvB gene is involved in the formation of a cyclic b-I ,2-glucan (Hooykaas & Schilperoot, 1992); the protein of the chvA gene, with extracellular polysaccharides (131). The ChvE protein interacts with the periplasmic domain of VirA protein to potentiate the response of this environmental sensor of the bacterial cell to the external phenolic inducer (B[acute{e}]langer et al., 1997; Dy[acute{e}] & Delmotte, 1997).

The T-DNA portion of the plasmid is converted into a single-strand (ss) copy, T-strand, as a transfer intermediate (Fig. 3). (Recently, some evidence of the transfer of double-stranded DNA was obtained in some bacterial lines [Steck, 1997]). This T-strand travels through prokaryotic and eukaryotic membranes and cellular compartments by means of association with certain Vir proteins; the T-strand bound with VirD2 and VirE2 is called the T-complex. This complex is targeted to the plant-cell nucleus, and the T-strand becomes integrated into a plant chromosome. The VirD2 protein (an endonuclease) cuts the T-DNA borders and becomes attached to the 5' end of the single-stranded T-DNA. The VirE2 protein then binds to the surface of this single-stranded T-DNA--VirD2 unit, protecting it from plant nucleases and forming the so-called T-complex (Cook et al., 1997; Tinland, 1996; Zupan et al., 1996; Zupan & Zambryski, 1997). Much of the detail of this transfer and subsequent function is unknown (Deng et al., 1999; Zhou & Christie, 1999). The transfer through the complex membrane channel between the bacterium and plant cell is believed to be mediated by VirB proteins (Dang et al., 1999; Lai & Kado, 1998; Zupan et al., 1999). The T-complex is then transferred through the nuclear pore, followed by integration of the T-DNA with the plant-cell genome. Little is known about this integration process, but it is an active area of research (Mysore, 2000).

Transcription occurs, and two major types of products, opines and the plant hormones IAA and cytokinin, are synthesized in the plant-cell cytoplasm upon translation by a series of genes in the T-DNA portion (labeled one genes in Hooykaas & Schilperoot, 1992). The IAA genes identified are tmsl and tms2 and ipt gene for cytokinin (Davey et al., 1994; Morris, 1986, 1995; Chou et al., 1998). The newly synthesized plant hormones start the numerous cell divisions to form the gall structure. Ethylene has also recently been shown to play a critical role in crown gall morphogenesis (Aloni et al., 1998). Tumor-induced ethylene substantially decreases the vessel diameter in the host tissues in wild-type stems but has a limited effect in the Nr mutant in tomato stems. Ethylene promotes the typical unorganized callus shape and surface of the gall. The opines, formed by genes in the T-DNA transferred, are then secreted from the plant cell and act as chemoattractants for agrobacteria. They serve as C and N sources for the bacterium attached to the plant cell, as well as for the surrounding aggregates of bacteria, by means of catabolic genes remaining in the bacterial plasmids (Kim & Farrand, 1996, 1998; Kim et al., 1996). Chemotaxis to opines may foster the interaction between agrobacteria and their plant hosts (Davey et al., 1994). Because opines are specific chemoattractants, they can play an important role in determining the distribution of agrobacteria in the tumor sphere, and they are the key chemical mediators that establish the Agrobacterium--plant interaction. A wide variety of opines and the genes for both catabolic and synthetic reactions have been identified. As diagrammed in Figure 4, the opine synthetic genes are found in the T-region of DNA of the plasmid and are transferred to the plant cell, whereas the catabolic opine genes are found in the tra system involved in conjugal transfer of plasmids to other bacteria (Cook et al., 1997; Farrand, 1998; Piper et al., 1999). The type of opine involved is specific for ea ch bacterial strain.

VI. Special Aspects of the Tumors


Relatively normal-appearing tobacco tumor shoots can be obtained by serially grafting tumor shoots to normal plants. Early experiments were done in the 1950s by Braun and other workers (Braun, 1982). By about 1976 Braun and Turgeon's group found that seeds of such grafts gave rise to normal plants and that recovery occurred during meiosis (Braun, 1965, 1982; Turgeon, 1982). Grafting is necessary because tumor shoots do not root; multiple rounds of grafting are needed to produce normal growth of the tumor shoot (Nester et al., 1984). The grafted shoots produced retain the tumorous properties of opine synthesis, such as nopaline, and phytohormone-autonomous growth when cell cultured.

Two types of reversions are involved (Drummond, 1979). Phenotypic reversion occurs when teratoma shoots (leafy tumors) are grafted to the apex of a normal plant, so that when forced into rapid growth they gradually regain normal morphology. However, the redifferentiated plant contains T-DNA, synthesizes opines, and reverts to a tumor phenotype when explanted back into tissue culture. This may mean that phenotypic reversion has an epigenetic basis. Genotypic reversion can also occur. The redifferentiated teratoma shoots sometimes flower and produce viable seed. Plants grown from these seeds lack T-DNA's or opines and require exogenous hormones in tissue culture. This apparent complete recovery occurs during meiosis. Haploidy, therefore, is apparently an important step, and diploid cells rarely recover completely. Three ways of recovery at the genotypic level are possible: haploid progeny with T-DNA are not viable; chromosomes with the T-DNA are specifically excluded; and unknown machinery in the genome can ex cise extraneous material. However, the exact mechanism of recovery is unknown,


Whereas tissue cultures of normal, healthy plant cells cultured in vitro need an exogenous supply of auxins and cytokinins as plant hormones for continued growth, some cells lose this requirement during subculturing and become able to grow on hormone-free media. The loss of an auxin requirement was identified in 1942 as habituation (Gautheret, 1955). It now is frequently noted in cultures of many species, and it can also involve cytokinins (Braun, 1978; Meins, 1989; Syono & Fujita, 1994; Chou et al., 1998). Habituation is often mentioned in the crown gall literature because of its striking resemblance to crown gall cell cultures that grow without the addition of exogenous hormones. Habituation, however, is a form of neoplastic transformation, in that it involves heritable, progressive changes in the cell phenotype to produce phytohormone-autonomous growth. No infectious agent has been identified, and the process is frequently reversible.


The host range of A. tumefaciens in crown gall tumor production includes a wide range of dicotyledonous plants mainly by phenolics in both herbaceous and woody plants, woody gymnosperms by means of resins and a phenolic glycoside (coniferin) in one case (184), and a few monocots (for example, asparagus, narcissus, gladiolus, yam) (Binns & Thomashow, 1988; Cervera et al., 1998; Conner & Dommisse, 1992; Huang et al., 1993; Schl[ddot{a}]ppi & Horn, 1992). The responses of both the host-specific bacterium and the plant are involved in the maintenance of such a range. No cell division occurs in wounds of most monocots, so that the wound response is inadequate. Instead, differentiation into lignified or sclerified cells occurs, forming a ring of hardened cells around the initial wound site. Asparagus in the Liliaceae can be transformed by Agrobacterium tumefaciens because it does have an adequate wound response, whereas most monocots do not (Binns & Thomashow, 1988). There also are few dicots that cannot be transf ormed easily (grain legumes) because they do not have an appropriate wound response (Potrykus, 1991). The production of phenolics as general defense products complicates the issue, and monocots differ in the distribution and type of phenolics in both the infected cells and the surrounding ones. Monocot walls are different in the phenolic cross-linkages between cell-wall components. High contents of feruloylated glucuronoarabinoxylans are present and are available for cross-linking by peroxidase wall enzymes that control the decline in cell expansion within an elongation zone (Bacon et al., 1998). The reason(s) for the lack of effective Agrobacterium-mediated transfer in monocots is obviously still not fully known.

Some evidence of T-DNA transfer has now been found in graminaceous monocots, such as maize, but only by the use of immature embryonic tissues (Schl[ddot{a}]ppi & Horn, 1992). Gene transfer into intact, transgenic rice cells by A. tumefaciens--mediated transformation has been achieved by coincubation of rice cells with Agrobacterium in the presence of a potato cell suspension culture rich in phenolics such as acetosyringone, by the construction of a superbinary vector with several vir genes and the use of a more virulent Agrobacterium strain (Chan et al., 1993). In another study with rice tissues cocultivated with A. tumerfaciens, the sequence analysis revealed that the T-DNA in the transgenic rice plants derived was essentially identical to those in transgenic dicotyledons and that the efficiency of transformation was similar to that with dicots; the techniques used in these studies permit Agrobacterium-mediated gene transfer into rice as a straightforward and routine method, and recently to a calcitrant var iety (Hiei et al., 1994; Mohanty et al., 1999).


A variety of terms have been used to describe the galls in natural plants and those under culture: tumorous, cancerous, neoplastic growths, oncogenic, malignancy. No plant tumors are known to metastasize (Kalil & Hildebrandt, 1981). The relatively rigid cell wall enclosing each plant cell upon cell division forms a barrier to any metastatic spread of crown gall within the host and, therefore, precludes the presence of a cancer s.str. (Lippincott & Lippincott, 1981). In other words, plant cells cannot migrate; the newly formed cell plate followed by the cell wall separates the new from the old. The term "gall" is also used to describe insect, mite, viral, and other induced growths (Kalil & Hildebrandt, 1981).

Comparisons have recently been made in roots of transgenic plants with the regulation of postembryonic organogenesis mediated by genes of meristems controlling progression through the cell cycle, such as cyc and cdc genes that control [beta]-cyclin (Doerner et al., 1996; Doonan & Hunt, 1996). The main argument given is that plants are not subject to cancerous growths, as animals are, because cell lineage plays the major role in determining the identity of animal cells, whereas extra cells produced in the root meristems of plants simply enlarge the pool of cells, but the cell identity in differentiated zones is fixed by its neighbors rather than by their genealogy.


Conjugation is a process in which there is a unidirectional transfer of genetic information through direct cellular contact between a donor and a recipient bacterial cell. The donor state is conferred by the presence of a plasmid factor. Conjugation results in the transfer of this genetic factor from donor to recipient, and the entire plasmid is transferred from the donor to the recipient. The possibility that the mechanism of transfer of DNA from the bacterium to the plant cell in crown gall disease is similar to the conjugative system in bacteria was noted in early studies (Binns & Thomashow, 1988). This similarity has been recently emphasized in genetic studies of the plasmids involved in both processes. Conjugal plasmids have now been well studied in Agrobacterium tumefaciens (Cook et al., 1997; Farrand, 1998; Kim & Farrand, 1998). The vir and T-regions of the vir system of DNA are clearly separated from two conjugal transfer regions (the tra system) involved in bacterial conjugation. As shown in Figure 4, two areas (clusters of genes) of classical Ti-plasmids, the tra system consisting of tra and trb regions, are involved in bacterial conjugation. Their known genetic structure has been recently reviewed (Farrand, 1998). TraA protein is the major structural subunit of the F pilus of A. tumefaciens, and VirB2 is a homolog of TraA. VirB2 protein is the major pilin subunit of the pilus that mediates the transfer of the T-DNA Agrobacterium to plants by lining the channel during the transfer from the bacterium to the plant cell (see Fig. 3) (Lai & Kado, 1998). Opines are key parts of this conjugation process between bacteria, as well as in the crown gall transformation. Genes involved in the synthesis and catabolism of opines are found on different portions of the bacterial plasmid. Whereas opine synthesis genes are transferred to the plant cell by means of the T-DNA portion of the plasmid, genes that confer catabolism of the opines found in the vir region of the bacterial plasmid are not transferred to the plant cell. The tra system, in other words, functions independently from the vir system, and conjugation does not require functions encoded by the vir system (Farrand, 1998). Coevolution of plasmidic and chromosomal sequences involving chvE and virA gene interaction in conjugative transfer of Ti-plasmids to a new host bacterium has recently been discussed (B[acute{e}]langer et al., 1997)

VII. Hairy Root Disease and Ti-Plasmids of A. rhizogenes

Hairy root disease, found mainly in dicotyledonous roots, is characterized by an abundant proliferation of roots at the site of infection by A. rhizogenes. As in the case of crown gall disease, there is a transfer of genetic material from a plasmid of the infecting organism, A. rhizogenes, that is stably integrated into the plant genome (Gelvin, 1990; Petersen et al., 1989; Schaerer & Pilet, 1993). The host range of A. rhizogenes is much more limited under natural conditions than is that of A. tumefaciens. Such infections have been found only in perennial dicotyledonous plants, not in annuals, and in species of Spiraea, aspen, and apple trees (Nilsson et al., 1997). The typical phenotype involves extensive lateral root proliferation, synthesis of opines, lack of a gravireaction, and an unusually high density of root hairs in phytohormone-free cell-culture media. The plasmids are classified according to the opines produced by transformed tissue (Petersen et al., 1989). Plasmids of the nopaline and octopine ty pe have a single-sized T-DNA portion essential for transfer. The nopaline or agropine T-DNA consists of two separated T-DNAs, a left ([T.sub.L]) and a right border ([T.sub.R]) (see Petersen et al., 1989: fig. 1). The two auxin biosynthetic gene, aux1 and aux2, are located on opposite DNA strands in the [T.sub.R] region in the agropine type strain (Gaudin & Fraley, 1993). Unlike crown gall disease, there is no de novo synthesis of IAA, although there are auxin genes with weak oncogenic activity in the [T.sub.R]-DNA portion of the octopine opine type. The major determinant in some cases involves a hypersensitivity to IAA (in the [T.sub.L]-DNA portion of an opaline type of opine) that produces the excessive proliferation of lateral roots, while using the limited amount of endogenous plant auxin (Petersen et al., 1989). The [T.sub.L]-DNA of the Ri-plasmid is the best-studied portion and contains the rot A, B, C, D genes that apparently affect this auxin-sensitivity process. These auxin effects, however, are varie d in different dicots and need further study. Earlier work indicated that the Rol proteins are involved in the regulation of planthormone metabolism, but recent data indicate that this is not the case (Nilsson et al., 1993, 1997; Nilsson & Olsson, 1997. The organization of Ti- (tumor-inducing) plasmids has been compared with that of Ri-plasmids (Gelvin, 1990; Petersen et al., 1989). The vir genes of the

Ri-plasmid appear to be similar to those of the Ti-plasmid. Many species can be easily regenerated from hairy roots when freed of the inciting bacteria, and they are maintained as cultures with or without added auxins and cytokinins, or as whole plants. However, they differ from normal plants in several ways (Gelvin, 1992). During the 1980s, Ri-plasmids of A. rhizogenes (disarming not required) and hairy root cultures for regeneration of intact plants were important as vectors for transgenic transformations, but disarmed binary vectors from A. tumefaciens have become much more useful. However, plasmids of A. rhizogenes and hairy root cultures are vital for studies of root development, physiology, enzymology, and the molecular genetics involved in transgenic plants (Christey, 1997). The genes rolB and rolC differ in their function to stimulate adventitious root induction in tobacco-leaf segments (Aoki & Syono, 1999). The Ri-system as a vector for transformation of other species is discussed in the next section .

VIII. Genetic Engineering and Formation of Transgenic Plants

A whole new area of research developed when the genetic transfer of DNA of the T-strand was discovered. Transfer of new genes to plants started in the 1980s with the introduction of foreign genes to tobacco by means of portions of the Ti-plasmid from A. tumefaciens (Davey et al., 1994; Draper & Scott, 1991; Gasser & Fraley, 1992; Hiei et al., 1994; Hooykaas & Schilperoot, 1992). Genetic transformation of plants can now be approached by a variety of other methods (Potrykus, 1991), including viral vectors, gene transfer to protoplasts, particle-gun bolistics, microinjection into proembryos, electroporation, and so forth (Christou, 1996). Plants have been transformed by the introduction of possibly useful foreign genes for horticultural or agricultural use, as well as to study the expression and function of cloned genes by means of cell cultures or intact plants (Klee & Lanahan, 1995; Klee et al., 1987).

Ti-plasmids served as gene vectors in early work, but either the use of wild-type Tiplasmids failed to produce shoots or regenerated abnormal shoots did not develop roots. The first attempts to transfer foreign DNA into plants using the whole Ti-plasmid as a vector were made in 1980-1983. More effective methods with smaller sets of chimaeric genes (with mixed characteristics), consisting of a suitable eukaryotic promoter, the gene sequence of interest, and a termination sequence, were then used (Davey et al., 1994). All vectors require a selectable marker that functions in Agrobacterium and a selectable marker for expression in plants. Subsequently, binary vectors were used in which the necessary components were in separate reconstructed plasmids, each containing the necessary transfer conjugal gene complex (tra) (see Lindsay & Jones, 1990: fig. 6.11). One reconstructed Ti-plasmid (binary T-DNA plasmid) contained only the vital left and right borders of the T-DNA (3' & 5' termination sequences that flank the T-DNA region), to which foreign genes, along with reporter (e.g. GUS) and selectable marker (e.g., kanamycin) genes, had been added. This T-DNA plasmid was subsequently introduced into an Agrobacterium strain harboring a "disarmed" Tiplasmid lacking the T-DNA region (and its oncogenes for hormones) but with an intact vir region acting in trans on the T-DNA carried on a separate plasmid (Hooykaas & Schilperoot, 1984). Preservation of the vir gene region is vital for success, but removal of bacterial oncogenicity genes (onc) controlling hormones is necessary to form such "disarmed" vectors. Binary transformation vectors with T-DNA borders can be replicated in Eseherichia coli or A. tumefaciens, but vectors are now usually engineered in E. coli and transferred by conjugation into Agrobacterium strains that contain a "disarmed" Ti-plasmid but with an intact vir region. The foreign DNA inserted into the binary T-DNA plasmid vector is transferred to plant cells by the activity of the vir region. The chv gene in th e bacterial chromosome is also required for transfer. Stability and expression of foreign genes in transgenic plants vary, but transmission to seed progeny can occur in a Mendelian manner with a high degree of meiotic stability, especially in dicots (Gasser & Fraley, 1992; Kim & Farrand, 1998). Construction of a genomic library of plasmids and other agents such as virus, detection of a specific gene within a library, isolation of a specific DNA fragment, and cloning in plasmids or bacteriophages, are briefly and effectively described in the volume on Plant Biotechnology in Agriculture by Lindsay and Jones (1990: chaps. 3, 6, and 10).

The Ri-plasmid system is useful as a vector for transformation of dicots in some cases because it does not need to be disarmed, as in the Ti-system, and it has been used to produce transgenic plants. There is a problem of altered phenotypes, but the segregation of T-DNAs at meiosis permits corrections (Christey, 1997). Whereas a wide range of hosts can be used in transgenic studies with A. tumefaciens, the host range of A. rhizogenes is much more limited under natural conditions. Such infections have been found only in perennial dicotyledonous plants, not in annuals, and in species of Spiraea, aspen, and apple trees (Nilsson & Olsson, 1997). As mentioned in the section on A. rhizogenes, binary vectors have become more widely used in the 1990s for transgenic studies of crop plants, except for genes functioning mainly in the root and the advantage of selection due to the appearance of hairy roots (Christey, 1997).

These transformations to transgenic plants have involved induction of herbicide resistance in crop plants, insect resistance, and virus resistance (Gasser & Fraley, 1992; Lindsay & Jones, 1990). The same system has been used to study the effect of antisense genes on natural systems and the effect of transfer of enzymes producing secondary compounds not found in some plants. A recent review of transgenic crop plants is that of Christey (1997).

IX. Conclusions

The study of crown gall disease in plant stems is important, not only because of its long history but also because the present knowledge of the disease is based on a large number of disciplines and organisms: microbiology (bacteriology), plant physiology, plant and bacterial biochemistry, genetics and molecular genetics, transgenic plants and crops, and disease and evolutionary history. Inoculation with A. rhizogenes has been useful as a means of inducing adventitious root formation in hard-to-root woody species (121). The research on crown gall, therefore, is an excellent example of the interactions among many disciplines in modem biology. The transfer of DNA from a prokaryote to a eukaryote by Agro bacterium tumefaciens and A. rhizogenes is so far unique. The evolution of the origin of T-DNA in the bacterial plasmids is unknown, but it is of considerable interest because the T-DNA encodes genes that are expressed in both eukaryotic and prokaryotic cells. The hormone involved, IAA, is produced from indole-3 -acetamide, and both IAA and cytokinins can be produced by agrobacteria in rich media containing tryptophan or tyrosine. Homology exists between the T-DNA of the virulence plasmid of A. rhizogenes and uninfected carrot and tobacco. It has been speculated that the bacterium may have captured plant genes, which it then reintroduces into the plant cell. However, because the comparable sequences may have been conserved in both bacteria and plants throughout evolution, more data are necessary in order to further these possibilities. Grown gall disease, still an unfinished story, is a unique case history, not only because of the study of the disease itself, involving DNA transport between kingdoms, but also because the knowledge derived from it led to the knowledge and benefits of transgenic transformation studies that have outnumbered those of the disease itself.

Crown gall and its related adventitious root disease are still an unfinished and fascinating story. For instance, why have dicotyledonous plants not evolved a defense mechanism against the attacks of A. tumefaciens?

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Disease in grapevines


Bacterium involved (1907)

Plant-hormone connection

Secondary tumors along stem


Tumors autonomous in dicots: cancer?

Tissue culture and grafts; no added auxin or cell-division factors

TIP (tumor-inducing principle) involved


DNA suspected as TIP, but data inadequate

Auxin (b-IAA) and cell-division factor in tumor tissues

Reversal of tumor; graft to normal plant via seed


Adherence of A. tumefaciens to wound

Opines in gall-tumor tissue cultures


Source of TIP: T-DNA portion of Ti-plasmid

Carbohydrate involvement in adherence of bacterium

Virulent strain classification based on opines

Recovery from tumorous state during meiosis


Molecular genetics; major advances

T-strand enters; bound to plant chromosome

Regions in plasmid involved: vir, T-DNA, opine and hormone synthesis, opine catabolism

Vir proteins of plasmid identified

Hormone synthesis of bacterial genes in plasmid

T-DNA transferred to plant nucleus

Bacterial type of conjugation suspected

First transgenic plants; Ti-plasmids as gene vector


T-DNA restriction maps

Protein of constitutive VirA gene upon phosphorylation responds chemotatically to inducing phenolics; monosaccharides also involved

Proteins of chv and vir genes studied

VirG protein activates all other vir genes

Functions of vir proteins B, D, and E outlined

Negative control of virA-virG regulatory genes by ros

Opine classification, catabolic loci, synthetic loci

Comparison of conjugal plasmids in Rhizobiaceae

Genetic engineering technology of crops


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Publication:The Botanical Review
Geographic Code:1USA
Date:Jan 1, 2000
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