PLANT BIOTECHNOLOGY ASSIGNMENT

On

Tumor Formation on Plants Using A.tumefaciens

Submitted byDarpan Raghav

INTRODUCTION

Tumors are one of the most widespread abnormalities of plant morphogenesis. However, the mechanisms of tumor formation are poorly studied. Analysis of tumor formation in plants is important for better understanding of the control of cell division and differentiation and for identifying new plant oncogenes. Formation of tumors in plants may be caused by several factors. First, tumors may result from infection from viruses, bacteria, arthropods, and worms. Tumors induced by Agrobacteria are the most common. Second, tumors may be formed on plants of particular genotypes. At the plant level, the plant tumors were described in Pisum sativum, Raphanus sativus and other species, and in inter specific hybrids of the genera Nicotiana, Brassica, Lycopersicon, etc. In addition, habituation of plant in vitro cultures to hormone autotrophic state may be attributed to genetic tumors.

AGROBACTERIUM INDUCED TUMORS

Agrobacterium tumefaciens causes tumor formation in plants. Plant signals induce in the bacteria the expression of a range of virulence (Vir) proteins and the formation of a type IV secretion system (T4SS). On attachment to plant cells, a transfer DNA (T-DNA) and Vir proteins are imported into the host cells through the bacterial T4SS. Through interaction with a number of host proteins, the Vir proteins suppress the host innate immune system and support the transfer, nuclear targeting, and integration of T-DNA into host cell chromosomes. Owing to extensive genetic analyses, the bacterial side of the plantAgrobacterium interaction is well understood. However, progress on the plant side has only been achieved recently, revealing a highly complex molecular choreography under the direction of the Vir proteins that impinge on multiple processes including transport, transcription, and chromosome status of their host cells. Agrobacterium species are known as the only organisms capable of inter kingdom gene transfer. This soil-borne Gram-negative bacterium is a broad-host range plant pathogen, which initiates tumor formation on most dicotyledonous and some monocotyledonous species. Such tumors do not require the continuous presence of the bacteria for proliferation, showing that the plant cells have been transformed genetically. The factors required for tumor formation are encoded on a large tumor-inducing (Ti) plasmid of virulent Agrobacterium strains. The Ti

plasmid also serves as a source for the transfer DNA (T-DNA), a DNA region that is imported into plant cells and integrated into the host chromosomal DNA resulting in genetic manipulation of the host. The expression of T-DNA-encoded bacterial genes in the host cell results in the production of enzymes that catalyze the synthesis of plant hormones, which are responsible for tumor growth and the formation of novel amino acid sugar conjugates, termed as opines. As opines can serve as carbon and sometimes nitrogen sources for Agrobacterium to the exclusion of most other microorganisms, they provide a selective advantage for this species. The capacity for gene transfer into plants has been used to develop Agrobacterium tumefaciens as a vector for genetic manipulation. Engineered DNA segments of interest, which are first cloned into the T-DNA region of disarmed plasmids, are then introduced into Agrobacterium and subsequently transferred into plants. From these disarmed plasmids, the genes responsible for tumorous growth have been removed, ensuring that the transformed cells can be regenerated into fertile plants that transmit the engineered DNA to their progeny. By these means, the host range of Agrobacterium has been extended to include other bacterial species as well as fungi and even some mammalian cells. The capacity for gene transfer into plants has been used to develop Agrobacterium tumefaciens as a vector for genetic manipulation. Engineered DNA segments of interest, which are first cloned into the T-DNA region of disarmed plasmids, are then introduced into Agrobacterium and subsequently transferred into plants. From these disarmed plasmids, the genes responsible for tumourous growth have

been removed, ensuring that the transformed cells can be regenerated into fertile plants that transmit the engineered DNA to their progeny. Agrobacterium mediated transformation serves as an important model system for studying host pathogen recognition and delivery of macromolecules into target cells. The interaction between Agrobacterium and plant cells can be divided into several steps: recognition, virulence (Vir) gene expression, attachment to the host cell, targeting of Vir factors and T-DNA into the host cell, and chromosomal T-DNA integration. On chemical recognition of plant-derived compounds,

Agrobacterium Vir gene expression is induced, which is followed by the physical interaction between bacterium and plant cells. Bacterial transfer machinery is subsequently produced and assembled to import the de novo produced T-DNA strand along with a number of Vir factors into the host cell. Once inside the plant cell, the T-DNA is translocated into the nucleus, in which it integrates into the host chromosome. On expression of T-DNA genes, plant cells are re-programmed for tumor growth and production of opines.

An overview of the Agrobacteriumplant interaction.

Steps Involved In the Agrobacterium Plant Interaction

Recognition of plant cells as host by Agrobacterium

Agrobacterium strains are widely distributed in the soil. Moreover, most isolates do not contain a Ti plasmid and are capable of living independently of a plant host. Yet, as tumor produced opines are a specific food source for Agrobacterium, the capacity of Agrobacterium strains to induce such tumors is a clear selective advantage. However, because plant transformation is a complex process and energetically demanding, Vir gene expression must be carefully regulated. The identification of vir genes, which are required for virulence, but lie outside the TDNA, was a major step towards understanding the transformation process. With the exception of virA and virG, the vir genes were found to be essentially silent unless the bacteria are cultured with plant cell. Although vir gene induction depends on molecules exuded by the plant, attachment to plant cells is necessary for transformation and is mediated by chromosomally encoded Agrobacterium genes. Thus, host recognition by Agrobacterium resulting in transformation is composed of two independent processes: Vir gene activation and attachment to the host cell.

Agrobacterium Vir gene expression

The vir gene activation by plant factors requires two genes, virA and virG, which are constitutively expressed at a basal level, but can become highly induced in a feed-forwards manner. The virA and virG genes encode a two-component phospho-relay system in which VirA is a membrane-bound sensor and VirG is the

intracellular response regulator. On signal sensing, the histidine kinase VirA activates VirG through transferring its phosphate to a particular aspartate of VirG, thereby activating VirG to function as a transcription factor. Phosphorylated VirG then binds at specific 12 bp DNA sequences of the vir gene promoters (vir boxes), thereby activating transcription.

Plant entry sites for Agrobacterium

In nature, Agrobacterium attacks mainly wounded tissue. A wound site may simply be a portal of entry, but other specific processes specifically occurring at these sites are likely to facilitate transformation: wound secreted compounds such as phenols and sugars induce vir gene expression. In addition, the latter act as chemotactic attractants of Agrobacterium. Thus, wound-specific features such as high activity of the phenylpropanoid pathway, low pH, and sugars associated with cell wall synthesis/wound repair correlate with enhanced transformation frequency and efficiency. Although transformation can also occur in unwounded plants with Agrobacterium cultures grown in pre-induction medium it seems that Agrobacterium has optimised the VirA/VirG system to respond to signals from wound sites. Cell division activity at the wound sites is thought to be equally important for transformation (Braun, 1952). However, cells in the root elongation zone were found to be the most highly transformable (Yi et al, 2002). Cells of this non-meristematic endoreduplication. zone are not undergoing a normal cell cycle, but

Import of Agrobacterium Vir factors into host cells

The A. tumefaciens virB-encoded T4SS transports substrates across the bacterial cell envelope. Certain C-terminal motifs were found to be required for the export of targeted substrates. These export signals mediate the interaction of substrates with the T4SS. The C-termini of VirF, VirE2, and VirE3 are sufficient to mediate transport of fusion proteins to plants. The minimal size of VirF required to direct protein translocation to plants is the C-terminal 10 amino acids, from which the minimal consensus sequence R-X (7)-R-X-R-X-R required for substrate secretion by the VirB complex could be derived. C-terminal fusions of VirE2 blocked its translocation to host cells. Accordingly, insertion of a FLAG tag at the C-terminus of VirE2, or truncation of the C-terminal 18 amino acids of VirE2, renders the protein nonfunctional in A. tumefaciens, while not affecting its capability to bind single-stranded DNA. However, overexpression of such VirE2 C-terminal mutant derivatives in transgenic plants confers susceptibility to transformation by an A. tumefaciens virE2-deficient strain, suggesting that the mutations disrupted a region of amino acids required for translocation, such as a secretion signal.

Host cell entry of Agrobacterium factors

It is presently unclear how the Vir proteins and the T-DNA protein complex traverse the host cell wall and membrane barriers. In T4SS-mediated plasmid transfer, the pilus enables the interaction between donor and recipient, followed by the fusion of outer membranes in a mating junction. The mechanism by which the transferred conjugal intermediate traverses the bacterial wall and inner membrane

is not known. Even less is known about VirBmediated transfer across host cell barriers. The enormous host range transformed by Agrobacterium suggests that the specificity of hostpathogen interaction required to breach the host cell wall and membrane barriers may be less important than expected.

Targeting of Agrobacterium T-DNA into the host cell nucleus

Once inside the plant cell, the T-DNA must find its way into the nucleus. Several Agrobacterium Vir proteins, as well as a number of plant proteins, seem to be involved in this process the proteins VirD2 and VirE2 contain plant-active nuclear localization signal (NLS) sequences. VirD2, which is covalently linked to the 50end of the T-DNA, contains two NLS regions, both of which can direct chimeric proteins to the nucleus. Sterical considerations suggest that the bipartite NLS in the carboxy-terminus of VirD2 might be biologically important for nuclear targeting of the T-DNA complex VirE2 protein contains two separate bipartite NLS regions that can target fusion reporter proteins to plant nuclei fluorescently labeled single stranded DNA coated with VirE2 and microinjected into plant cells localizes to the nucleus, whereas naked single-stranded DNA remains in the cytoplasm.

Diagram showing the tumour formation and the host response.

Plant factors and defense responses involved in Agrobacterium tumour formation.

Although certainly unintentionally, the host plant actively participates in Agrobacterium transformation. This assistance occurs at several levels: Vir protein/T-DNA import, dissociation of the Vir/T-DNA complex, T-DNA integration, and re-programming of gene expression for tumour development. A number of host factors that are exploited by Agrobacterium to achieve transformation have been identified. Major progress has been made through yeasttwo-hybrid (Y2H) screens for identifying host proteins that interact with

Agrobacterium Vir proteins. Another important progress was achieved through large plant mutant screens.

TRANSFORMATION OF ZEA MAYS

Following steps are involved in the tumour formation of Zea Mays using A.tumefacians.

Preparation of Plant Material

The hybrid Zea mays L. variety Funk's G90 was used because of local availability of commercially processed seed and low incidence of seed-borne contamination after surface sterilization. To our knowledge, this hybrid is not one of the genotypes that are noted to be regenerable in vitro from protoplasts or callus. Seeds

were rinsed 15 min in running tap water, surface-sterilized in 20% Clorox for 15 min, rinsed three times with autoclaved water, allowed to imbibe water 2 to 4 h, and placed on agar-solidified medium (1% w/v) (pH 5.7), containing lx MS halide stock (23), to germinate. Seeds were incubated at 30C in the dark 1 to 3 days.

Thin section of corn apex which includes meristem and expanding leaves.

Isolation of the Shoot Apex

Shoot apices were removed from the germinating embryo or seedling. Care was taken to first isolate the apex from the embryo, followed by removal of tissue proximal to the base of the meristem region. Primordial and elongating leaves were not removed (Fig. 1). The outer dimensions of the isolated apex ranged from approximately 1.0 x 0.3 mm. Because the position of the apical meristem could not be seen directly during isolation, the size range of the explants is approximate.

Bacterial Strain, Plasmid, Culture Conditions

Agrobacterium tumefaciens EHA1, a strain derived from A281 containing the supervirulent pTiBO542 plasmid (16), and the binary Ti construct, pGUS 3. The construction was a HindIll to EcoRI fragment from pGUS (20) placed in pARC 8 (26). This fragment contained CaMV 35S fused with the gene coding for GUS2 (20) and a nopaline synthase polyadenylation site (NOS-TER). The construction ofpARC8 containing the chimeric gene NOS/NPT from pNEO105 and pARC4 was described by Simpson and colleagues (26). This construct was designed for efficient expression in tobacco and petunia but was employed because of availability in EHA 1. The efficiency of expression of pGUS3 in monocot tissues was unknown at the time. Agrobacteria were grown on agar solidified LB media containing 5 mg/L tetracycline.

Co cultivation and Induction of Agrobacteria

After 3 to 5 d of growth on LB media containing tetracycline, bacteria were scraped from the culture plate and a slurry was made using 0.5 mL aqueous solution of 10 mM nopaline (30) and 30 jLM acetosyringone (27). The bacterial suspension was applied directly to the cut base ofthe shoot apex. A single invasion into the meristematic region through the inoculated shoot base with a hypodermic needle (255/8 gauge) was performed to place bacteria in the shoot meristem. Induction of the bacteria was allowed to occur during the 2 d of contact with the plant tissue.

Plant Tissue Culture Media and Culture Conditions

The inoculated shoot apices were cultured on a basal medium containing the MS salt formulation and the following in mg/L: 0.1, kinetin; 100, m-inositol; 40, thiamine-HCl; 15,000, sucrose; 8,000, TC agar, pH 5.7. Kinetin was included in the initial culture medium only. At specific stages in the reculture cycle, supplements to this basal medium included the following in mg/L: 7.5, kanamycin; 500, carbenicillin. Media were sterilized by autoclaving 25 min under standard conditions and dispensed into 100 x 20 mm sterile, polystyrene petri plates at 25 mL/plate; 10 to 15 explants were cultured per plate. Cultures were wrapped with parafilm, incubated under a combination of Gro-lux (GE) lights and full-spectrum Vita-lite (Duro-test), 60 to 80,qE. m-2 s-', 16-h day at room temperatures ranging from 22 to 25C night/26 to 28C day.

Transfer of Plants from Tissue Culture

Growing conditions were as follows. Plants were transferred from culture to 1 gallon pots containing Metro-mix 352 (Terra-lite), fertilized with Peat-Lite Special 15-16-17 (Peters). Plants were grown under a 16-h d regime offull spectrum fluorescent Vita-lites (Duro-test Co.) (500-700 .E. m-2 s-') or metal halide high intensity lamps (900-1100 gE m-2 s-'), with little natural light supplementation. Temperatures ranged from 22 to 28C. These lighting conditions were minimal for corn but allowed the regenerated plants to grow 3 to 5 feet and flower. Male flowers matured 2 to 3 weeks before the female flowers were receptive, therefore self-pollination was impossible. Pollination was achieved using fresh pollen from available sources.

Germination of Progeny

The F, seed of one plant designated C, were disinfected as described previously. Embryos were removed from the seed to break dormancy and germinated in vitro using the hormone- free basal MS medium described. In vitro germination of progeny embryos was not necessary if senescing plants bearing seed were allowed to dry. This was the case for plant designated as C56. Seedlings were transferred to pots and grown as described. Due to plant crowding and relatively low light intensity, many of the ears of these plants were empty of seed; however, F3 generations have been obtained from C 1, and F2 generations from C56. None of these plants could be self-pollinated because of the wide difference in maturity of tassels and ears under the existing conditions.

Identification of Transformed Plants

Putatively transformed plants were identified on the basis of the fluorescent GUS assay (20). Chloramphenicol (20 mg/L) was added to the assay buffer and assays were incubated overnight. Regenerated plants were assayed for presence of GUS activity. Leaves from different areas of these primary transformants were assayed because of the possibility that the plants were chimeric for the transferred traits. All surviving regenerated plants were hand pollinated. The progeny were assayed for GUS. Assays were not corrected for protein concentration; however, uniformly sized tissue samples were used. Assays of the leaf tissues from the Fl, F2, and F3 plants were also performed. Plants which did not appear to have GUS were discarded.

BIBLIOGRAPHY
LITERATURE CITED
1. Binns AN, Tomashow MF (1988) Cell biology of Agrobacteriulm infection and transformation of plants. Annu Rev Microbiol 42: 575-606 2. Bytebier B, Deboeck F, Greve HD, Van Montagu M, Hernalsteens JP (1987) T-DNA organization in tumor cultures and transgenic plants of the

monocotyledon Asparagus officinalis. Proc Natl Acad Sci USA 84: 53455349

3. Christou P, Platt SG, Ackerman MC (1986) Opine synthesis in wild-type plant tissue. Plant Physiol 82: 218-221 4. Dale PJ, Marks MS, Brown MH, Woolston CJ, Gunn HV, Mullineaux PM, Lewis DM, Kemp JM, Chen DF, Gimoour DM, Flavell RB (1989) Agroinfection of wheat: inoculation of in vitro grown seedlings and embryos. Plant Sci 63: 237-245

5. DeCleene M (1985) the susceptibility of monocotyledons to Agrobacterium tumefaciens. Phytopathol Z 113: 81-89

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