DOCSLIB.ORG

Agrobacterium Ti Plasmid Ptia6 Encodes an Enzyme That Catalyzes Synthesis of Indoleacetic Acid (Auxin/Indoleacetamide/Oncogenes) LINDA S

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Agrobacterium Ti Plasmid Ptia6 Encodes an Enzyme That Catalyzes Synthesis of Indoleacetic Acid (Auxin/Indoleacetamide/Oncogenes) LINDA S Proc. Natl. Acad. Sci. USA Vol. 81, pp. 5071-5075, August 1984 Biochemistry Crown gall oncogenesis: Evidence that a T-DNA gene from the Agrobacterium Ti plasmid pTiA6 encodes an enzyme that catalyzes synthesis of indoleacetic acid (auxin/indoleacetamide/oncogenes) LINDA S. THOMASHOW*, SUZANNE REEVES+, AND MICHAEL F. THOMASHOW* *Bacteriology and Public Health and tProgram in Genetics and Cell Biology, Washington State University, Pullman, WA 99164-4340 Communicated by Arthur Kelman, April 30, 1984 ABSTRACT Stable incorporation of tumor-inducing (Ti) the pTiA6 transcript 2 gene in auxin independence. The nu- plasmid sequences, the T-DNA, into the genomes of dicotyle- cleotide sequence of the gene has been determined (22, 23). donous plants results in the formation of crown gall tumors. The results indicate that there is a 1404-base-pair open read- Previous genetic studies have suggested that the products of ing frame that could code for a polypeptide of Mr 49,800, a the genes encoding transcripts 1 and 2, which are encoded by value in agreement with the results of Schroder et al. (21). the TL-DNA region of pTiA6, are responsible for inducing the We now have constructed a plasmid that produces the tran- auxin-independent phenotype of crown gall tissues. Here we script 2 gene product at detectable levels in E. coli. Bacterial report the construction of a plasmid, pMTlacT2, which directs extracts containing this protein can convert indoleacetamide the synthesis of the Mr 49,800 polypeptide encoded by the tran- to indoleacetic acid, the natural auxin of plants. script 2 gene. Cell-free extracts prepared from Escherichia coli harboring this plasmid converted indoleacetamide to indole- MATERIALS AND METHODS acetic acid, the natural auxin of plants; extracts prepared from Bacterial Strains. E. coli strains MH3000 [araD139 A(ara, plasmidless strains of E. coli or strains harboring the cloning leu)7697 A(lac)X74 galU galK rpsL (Strr) ompR101] and vehicle pBR322 did not carry out this reaction. We conclude TK1046 [araD.139 A(argF-lac)U169 rpsL150 (Strr) relAl that the transcript 2 gene of pTiA6 codes for an enzyme that flbB5301 deoCi ptsF25 malPQ: :TnS ompBcsl] were ob- participates in auxin biosynthesis, probably an indoleaceta- tained from G. Weinstock and used as described (24) to con- mide hydrolase. struct LacZ+ clones and produce high levels of,galacto- sidase fusion proteins. E. coli MC1061 [araD139 A(ara, leu)7697 A(lac)X74 galU galK hsdR(r-) rpsL (Strr)] was ob- Agrobacterium tumefaciens can infect wound sites on a wide tained from M. Casadaban (University of Chicago) and used range of dicotyledonous plants and cause the formation of to construct pMTlacT2. E. coli SG20251 [araD139 A(lacIPO- crown gall tumors (for reviews, see refs. 1-3). The disease ZYA)U169 A(lon)100 rpsL (Strr) thi cps3::TnlO] was ob- process is characterized by a virulence mechanism that is tained from S. Gottesman (National Institutes of Health) and unique among described procaryotic-eucaryotic cell-cell 2 interactions: it involves the transfer of genetic information used for expression of the cloned transcript gene. from bacterium to plant. All virulent A. tumefaciens strains Plasmids. The open reading frame vehicle pORF2 (24) was harbor one of a diverse group of tumor-inducing (Ti) plas- provided by G. Weinstock. This vector (see Fig. 1) contains mids. During the course of infection, a portion of the Ti plas- the promoter, ribosome binding site, and the first 33 codons mid, the T-DNA, is stably transferred to the plant cells of the E. coli ompF gene fused out of frame to the ninth where it becomes integrated into the nuclear genome (4-10). codon of the lacZ gene of E. coli. There are restriction sites Expression of specific genes encoded by the T-DNA (11-17) at the point of the ompF-lacZ fusion into which DNA seg- causes an alteration in the normal metabolism of auxins and ments can be inserted. If the DNA segment has an open cytokinins, two classes of plant hormones that have key reading frame and if the insert puts the ompF and lacZ se- roles in controlling plant cell growth and development. quences into frame, a multihybrid protein having 03-galacto- Whereas normal plant tissues generally require both of these sidase activity is synthesized. pGL101 (25) was from G. phytohormones for in vitro propagation, crown gall tissues Lauer. This plasmid contains a "portable" promoter-i.e., can be propagated indefinitely without them (18-20). Three the ribosome binding sequences and promoter-operator re- genes encoded by the TL-DNA of octopine-type Ti plasmids gion of the E. coli lac operon. pMT40 contains the HindIII such as pTiA6 are believed to be responsible for this phyto- fragment X of pTiA6 (7) inserted into the HindIII site of hormone-independent phenotype: genes encoding tran- pBR325. To construct pMTaT2 (Fig. 1), this T-DNA frag- Willmitzer et al. are ment was ligated into the HindIII site of pAC1 (26). This scripts 1 and 2, as numbered by (14), latter plasmid is a pBR322 derivative that contains tandem involved in auxin independence; and the transcript 4 gene lac UV5 promoters (including the first eight codons of lacZ) has a role in cytokinin independence (11-15). Protein prod- in the EcoRI site and gives increased expression of the tetra- ucts from these genes have not yet been detected in tumor cycline-resistance gene. However, transcript 2 gene expres- tissues. However, Schroder et al. (21) have shown that they sion is not significantly enhanced in this vehicle (see text). are expressed at low levels in Escherichia coli minicells or in Media and Reagents. LB broth was as described (27). For coupled in vitro transcription-translation systems prepared plasmid maintenance, ampicillin was included at 150 ,ug/ml from either E. coli or A. tumefaciens. Their data indicate in plates and liquid media. LacZ+ clones were detected on that transcripts 1, 2, and 4 encode Mr 74,000, Mr 49,000, and plates containing 40 ,ug of 5-bromo-4-chloro-3-indolyl-p3-D- Mr 29,000 polypeptides, respectively. galactoside per ml. We have focused our efforts on understanding the role of DNA Manipulations. Restriction enzymes, BAL-31 exonu- clease and T4 DNA ligase were from New England Biolabs The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: Ti plasmid, tumor-inducing plasmid; T-DNA, Ti in accordance with 18 U.S.C. §1734 solely to indicate this fact. plasmid DNA sequences stably transferred to the plant. 5071 Downloaded by guest on September 26, 2021 5072 Biochemistry: Thomashow et al. Proc. NatL Acad Sci. USA 81 (1984) FIG. 1. Construction of plasmids that code for an ompF-transcript 2-lacZ gene fusion (pLTt2-6) and synthesis of the transcript 2 M, 49,800 protein (pMTlacT2). In step 1, recombinant molecules that coded for the production of OmpF-transcript 2-LacZ proteins were constructed by digesting pMT40 with Bgl II, treating the DNA fragments with BAL-31 and inserting them into the Sma I site of pORF2. These molecules were then transformed into E. coli MH3000, and colonies were screened for f-galactosidase production on plates containing 5-bromo-4-chloro-3- indolyl-f3-D-galactoside. Plasmid pLTt2-6 contained a transcript 2 gene fragment of about 750 base pairs with the same 5'-to-3' orientation as the ompF and lacZ genes. Step 2 shows the construction of pMTaT2Z, a plasmid containing tandem lac promoters upstream ofthe transcript 2-lacZ gene fusion. pLTt2-6 and pMTaT2 were digested with Pst I and Sma I, mixed, ligated, and used to transform E. coli MC1061. E. coli MC1061 harboring pMTaT2Z produced 50 units of /3-galactosidase activity, whereas E. coli with a fully induced lac operon produced about 1000 to 2000 units of the activity. Step 3 shows placement of a lac "portable" promoter upstream to the transcript 2-lacZ gene fusion. pGL101 was digested with Pst I and Pvu II, and the DNA fragment containing the lac promoter and ribosome binding site was gel-purified. pMTaT2Z was linearized by HindIII digestion, treated with BAL-31 for varying amounts of time, digested with Pst I, and the large DNA fragments containing the transcript 2 open reading frame fused to lacZ were gel purified. This DNA was then mixed with the lac promoter fragment, ligated, and used to transform E. coli MC1061. Transformants were identified using indicator plates as before. The bluest colonies were picked and assayed for 3- galactosidase activity. E. coli MC1061 harboring pMTlacT2Z-12 produced 5000 to 6000 units of ,B-galactosidase activity and synthesized a novel protein ofabout M, 153,000, the approximate size expected for the transcript 2-LacZ fusion protein (data not shown). Step 4 shows construction of pMTlacT2, a plasmid producing the Mr 49,800 transcript 2 gene product. pMTaT2 was digested with Pst I and Sma I, and the DNA fragments containing the 3' end of the transcript 2 open reading frame were gel-purified. Plasmid pMTlacT2Z-12 was digested with Pst I and Sma I, mixed with the purified pMTaT2 fragments, ligated, and used to transform E. coli MC1061. Plasmid DNA was isolated from a number of white transformants on the indicator plates, and the desired plasmid, pMTlacT2, was identified by restriction enzyme analysis. It contained the transcript 2 gene preceded by the lac promoter and ribosome binding site. bla, 3-lactamase; tet, tetracycline resistance; P, Pst I; E, EcoRI; B, BamHI; S, Sma I; H, HindIII; PV, Pvu II; lacPSD, the lac promoter and ribosome binding site; lacP, the tandem lac promoters from pAC1.
Load more

Recommended publications
  • Co-Integrated Vectors
    Co-integrated Vectors • This vectors were among the first types of modified and engineered Ti plasmids devised for Agrobacterium - mediated transformation, but are not widely used today. • These vectors are constructed by homologous recombination of a bacterial plasmid with the T-DNA region of an endogenous Ti plasmid in Agrobacterium. Integration of the two plasmids requires a region of homology present in both. • Three vectors are necessary in this system: • Disarmed Agrobacterium Ti plasmids In these Ti plasmids, the oncogenes located in the T-DNA region have been replaced by exogenous DNA. Examples of these vectors include: – pGV series: the phytohormone genes are excised and substituted by part of pBR322 vector sequence. The left and right border sequences as well as the nopaline synthase gene of the Ti plasmid are conserved. • Intermediate vectors These are small pBR322-based plasmids (E. coli vectors) containing a T-DNA region. They are used to overcome the problems derived from the large size of disarmed Ti plasmids and their lack of unique restriction sites. Intermediate vectors are replicated in E.coli and are transferred into Agrobacterium by conjugation. They cannot replicate in A. tumefaciens and therefore, carry DNA segments homologous to the disarmed T-DNA to permit recombination to form a co-integrated T-DNA structure. • Helper vectors These are small plasmids maintained in E. coli that contain transfer (tra) and mobilization (mob) genes, which allow the transfer of the conjugation-deficient intermediate vectors into Agrobacterium. A resulting co-integrated plasmid assembled by in vitro manipulation normally contains: the vir genes, the left and right T-DNA borders, an exogenous DNA sequence between the two T-DNA borders, and plant and bacterial selectable markers.
    [Show full text]
  • Genetic Transformation of Hela Cells by Agrobacterium
    Genetic transformation of HeLa cells by Agrobacterium Talya Kunik*†‡, Tzvi Tzfira*, Yoram Kapulnik†, Yedidya Gafni†, Colin Dingwall‡§, and Vitaly Citovsky*¶ *Department of Biochemistry and Cell Biology, State University of New York, Stony Brook, NY 11794-5215; †Institute of Field and Garden Crops, Agricultural Research Organization, P.O. Box 6, Bet-Dagan 50250, Israel; and ‡Department of Pharmacology, State University of New York, Stony Brook, NY 11794-8651 Edited by Eugene W. Nester, University of Washington, Seattle, WA, and approved December 8, 2000 (received for review July 13, 2000) Agrobacterium tumefaciens is a soil phytopathogen that elicits the control of the simian-virus-40 early promoter and Herpes- neoplastic growths on the host plant species. In nature, however, simplex virus thymidine kinase polyadenylation signal (derived Agrobacterium also may encounter organisms belonging to other from pEGFP-C1, CLONTECH) subcloned into the Klenow- kingdoms such as insects and animals that feed on the infected blunted EcoRI and HindIII sites of the pPZP221-based binary plants. Can Agrobacterium, then, also infect animal cells? Here, we vector (15), between its right and left nopaline-type T-DNA report that Agrobacterium attaches to and genetically transforms borders. several types of human cells. In stably transformed HeLa cells, the integration event occurred at the right border of the tumor- Bacterial Strains and Host Cell Lines. A. tumefaciens-strain C58C1, inducing plasmid’s transferred-DNA (T-DNA), suggesting bona fide harboring Ti-plasmid pGV3850 (16), was used as host for the T-DNA transfer and lending support to the notion that Agrobac- pNeo binary vector. Strain C58NT1, which does not contain a Ti terium transforms human cells by a mechanism similar to that plasmid (17), was used as control.
    [Show full text]
  • Plant Virus DNA Replication Processes in Agrobacterium
    Proc. Natl. Acad. Sci. USA Vol. 93, pp. 10280-10284, September 1996 Evolution Plant virus DNA replication processes in Agrobacterium: Insight into the origins of geminiviruses? (tomato leaf curl virus/rolling-circle replication/molecular evolution) J. E. RIGDEN*, I. B. DRY, L. R. KRAKE, AND M. A. REZAIANt Division of Horticulture, Commonwealth Scientific and Industrial Research Organization, GPO Box 350, Adelaide 5001, Australia Communicated by Allen Kerr, Adelaide, Australia, July 1, 1996 (received for review May 29, 1996) ABSTRACT Agrobacterium tumefaciens, a bacterial plant the accumulation of these infectious, monomeric viral DNA pathogen, when transformed with plasmid constructs con- species within the bacterial cell. taining greater than unit length DNA of tomato leaf curl geminivirus accumulates viral replicative form DNAs indis- tinguishable from those produced in infected plants. The MATERIALS AND METHODS accumulation of the viral DNA species depends on the pres- Construction of Clones. TLCV ORFs are present on both ence of two origins of replication in the DNA constructs and the virion-sense (encapsidated strand) and the complementary is drastically reduced by introducing mutations into the viral strand of the viral ds replicative form (RF) as shown in Fig. 1. replication-associated protein (Rep or Cl) ORF, indicating Construction of virion-sense ORF mutants is described in that an active viral replication process is occurring in the detail in Rigden et al. (8). These include frame-shift mutations bacterial cell. The accumulation of these viral DNA species is in the Vl ORF at nucleotide position 153 (Vi mutant), in the not affected by mutations or deletions in the other viral open V2 ORF at position 663 (V2 mutant) or in both (V1/V2 reading frames.
    [Show full text]
  • TRANSGENIC PLANTS: Creation
    TRANSGENIC PLANTS: Creation Transgenic plants can be produced by Microprojectile bombardment: shooting DNA-coated tungsten or gold particles into plant cells. Electroporation: use of a short burst of electricity to get the DNA into cells. Agrobacterium tumefaciens-mediated transformation: The plant cells are totipotent, therefore a genetically modified single cell can regenerate into a new plant. A. tumefaciens is a soil bacterium responsible for crown gall disease of dicotyledonous plants. The galls or tumors are formed at the junction between the root and the stem of infected plants. After the infection by A. tumefaciens, the plant cells begin to proliferate and form tumors, and they begin to synthesize an arginine derivative called opine. The opines synthesized are usually either nopaline or octopine depending on the strain of A. tumefaciens. These opines are used as energy sources by the infecting bacteria. The ability of A. tumefaciens to induce crown galls in plants is controlled by genetic information carried on Ti plasmid (tumor-inducing plasmid). Ti plasmid has two components the T-DNA (Transferred DNA) and the vir region, which are essential for the transformation of plant cells. During the transformation process, the T-DNA is excised from the Ti plasmid, transferred to a plant cell, and integrated into the DNA of the plant cell. The integration of the T-DNA occurs at random chromosomal sites. In some cases, multiple T-DNA integration events occur in the same cell. Structure of Ti plasmid Structure of the nopaline Ti plasmid pTi C58: ori, origin of replication; Tum, genes responsible for tumor formation;Nos, genes involved in nopaline biosynthesis; Noc, genes involved in the catabolism of nopaline; vir, virulence genes.
    [Show full text]
  • Role of T-DNA Borders in the Transfer Process (Nopaline/Plants/Gene Transfer)
    Proc. Natl. Acad. Sci. USA Vol. 83, pp. 4428-4432, June 1986 Genetics Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: Role of T-DNA borders in the transfer process (nopaline/plants/gene transfer) R. B. HORSCH AND H. J. KLEE Monsanto Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198 Communicated by Howard A. Schneiderman, February 24, 1986 ABSTRACT We have developed a sensitive leaf disc trans- These data suggest that either transfer or integration is being formation procedure for studying early and/or transient T- directed in a polar fashion from the right border sequence. It DNA expression during Agrobacterium tumefaciens-mediated should be noted that none of these experiments can distin- transformation of plant cells. Using this system, we have guish between transfer and integration, since formation of a examined the function of T-DNA border sequences on the early tumor requires both processes to occur. expression of T-DNA genes and on the stable integration of We have developed a system to measure early expression those genes in infected cells. Deletion of the right border from of T-DNA genes in a plant cell and subsequent stable the T-DNA appears to permit transfer ofT-DNA genes from the integration of those genes. Plant cells are cocultured with A. tumor-inducing (Ti) plasmid but greatly reduces the frequency tumefaciens carrying a T-DNA with the genes for nopaline of their stable integration. A binary vector has been construct- synthase (NOS) and a selectable marker encoding kanamycin ed to permit examination of T-DNA border function in trans to resistance (NOS-NPTII-NOS) (24).
    [Show full text]
  • Ti PLASMID CODED PROTEIN in AGROBACTERIUM TUMEFACIENS
    AN ABSTRACT OF THE THESIS OF. Michael Maurice Engelgau for the degree of Master of Science in Biochemistry and Biophysics presented on September 5, 1979 Title: Ti PLASMID CODED PROTEIN IN AGROBACTERIUM TUMEFACIENS Abstract approved: Redacted for privacy I Robert Richard Becker Agrobacterium tumefaciens is a plant pathogenic bacteria which in- cites crown gall tumors. Although virulence has been associated with a 97-156 Mdalton "tumor-inducing" (Ti) plasmid, few potential plasmid gene products have been identified. The objective of this study was to identify plasmid-coded gene products through a comparison of protein fingerprints of virulent and avirulent isogenic strains. This required development of techniques for cell fractionation which could provide comparable protein samples from the various strains of bacteria and a reproducible high resolution method of protein characterization. The analytical system developed consisted of isoelectric focusing in the first dimension followed by SDS-polyacrylamide gel electrophoresis in the second dimension and was capable of reproducibly resolving nearly 500 proteins using this two-dimensional system. A. tumefaciens strain C58, which contains a nopaline Ti plasmid, produced five proteins detected neither in plasmid-free isogenic strains nor in A277. Strain A277, which contains the octopine plasmid, pTiB6 -806, produced three proteins detected neither in plasmid-free isogenic strains nor in C58. Strain A136, a plasmid-free derivative of C58, was deficient in two proteins found in the other three
    [Show full text]
  • T-DNA from Agrobacterium Ti Plasmid Is in the Nuclear
    Proc. Nati. Acad. Sci. USA Vol. 77, No. 7, pp. 4060-4064, July 1980 Botany T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells (plant tumors/teratomas/fragmentation patterns/chloroplast DNA/mnitochondrial DNA) MARY-DELL CHILTON*t, RANDALL K. SAIKI**, NARENDRA YADAVt, MILTON P. GORDON§, AND FRANCIS QUETIERI Department of Microbiology and Immunology and #Department of Biochemistry, University of Washington, Seattle, Washington 98195; tDepartment of Biology, Washington University, St. Louis, Missouri 63130; and ILaboratoire de Biologie Moleculaire Vegetale, Universit6 Paris-Sud, 91405 Orsay, France Communicated by Armin C. Braun, April 28, 1980 ABSTRACT The crown gall teratosa tumor line BT37, in- present in normal Parthenocissus tricuspidata mitochondrial cited by Agrobacterium tumefaciens strain T37,1has been found DNA (18). to contain part of the tumor-inducing plasmid, Ti T37, of the A final argument supporting the view that either chloroplast inciting strain. This foreign DNA segment, called T-DNA, is maintained at several copies per diplroid tumor cell. We have or mitochondrial DNA might harbor the T-DNA is the evidence examined subcellular DNA fractions from this tumor line in an that T-DNA can be lost from crown gall tumor cells. Braun and effort to determine whether T-DNA is in chloroplasts, mito- his collaborators (19, 20) showed that cloned crown gall tera- chondria, or nuclei. Tumor cell chloroplast DNA exhibited toma tissue could, when grafted to the apex of a decapitated EcoRI and Bst I endonuclease cleavage patterns identical to tobacco plant, give rise to normal-appearing shoots (19, 20), one those of normal tobacco chloroslast DNA.
    [Show full text]
  • The Expression of Foreign Gene Under the Control of Cauliflower Mosaic Virus 35S RNA Promoter
    Cell Research (1990), 1, 1-10 The expression of foreign gene under the control of cauliflower mosaic virus 35s RNA promoter Wang Hao and Bai Yongyan Shanghai Institute of Plant Physiology, Academia Sinica. 300 Fenglin Road, Shanghai 200032, China ABSTRACT The promoter region of cauliflower mosaic virus (CaMV) 35s RNA was employed to construct an intermediate expres- sion vector which can be used in Ti plasmid system of Agro- bacterium tumefaciens. The original plasmid, which contains a polylinker between CaMV 35s RNA and its 3' termination signal in pUC18 was modified to have another antibiotic resistance marker (kanamycin resistance gene Kmr) to facili- tate the selection of recombinant with Ti plasmid. Octopine synthase (ocs) structural gene was inserted into this vector downstream of CaMV 35s RNA promoter. This chimaeric gene was introduced into integrative Ti plasmid vector pGV3850, and then transformed into Nicotiana tobaccum cells. A binary plasmid vector was also used to introduce the chimaeric gene into tobacco cells. In both cases, the expression of ocs gene was demonstrated. The amount of oc- topine was much more than the nopaline synthesized by no- paline synthase (nos) gene transferred at the same time with Ti plasmid vector. This demonstrated that CaMV 35s RNA promoter is stronger in transcriptional function than the pro- moter of nos in tobacco cells. Key words: Agrobacterium tumefaciens, gene expression, cauliflower mosaic Cirus 35s RNA promoter. INTRODUCTION During the past decade, extensive research on the Ti plasmid of Agrobacterium tumefaciens has brought development of useful and efficient vectors to introduce fo- reign genes into dicotyledonous plants.
    [Show full text]
  • Methods of Gene Transfer in Plants A) Vector-Mediated Gene Transfer: (Agrobacterium-Mediated Gene Transfer)
    Methods of Gene Transfer in Plants Transgenic plants are those plants in which foreign genes have been introduced and stably integrated into the host DNA. It results in the synthesis of appropriate gene product by the transformed plants. The different methods of introducing foreign DNA into the plant genome have been grouped under two broad categories: A) Vector-mediated gene transfer and B) Direct gene transfer. A) Vector-mediated gene transfer: (Agrobacterium-mediated gene transfer) Agrobacterium tumefaciens and Agrobacterium rhizogenes are soil-borne, Gram-negative bacteria. These are phytopathogens (that cause infection in plants) and are treated as the nature’s most effective plant genetic engineer. A. tumefaciens induces crown gall disease and A. rhizogenes that induces hairy root disease in plants. Crown Gall Disease- Ti plasmid Almost 100 years ago (1907), Smith and Townsend postulated that a bacterium was the causative agent of crown gall tumors, although its importance was recognized much later. As A. tumefaciens infects wounded or damaged plant tissues, it induces the formation of a plant tumor called crown gall. The entry of the bacterium into the plant tissues is facilitated by the release of certain phenolic compounds (acetosyringone, hydroxyacetosyringone) by the wounded sites. Formation of a Crown Gall Tumor Crown gall formation occurs when the bacterium releases its Ti plasmid (Tumor- inducing plasmid) into the plant cell cytoplasm. A fragment of Ti plasmid, referred to as T-DNA, is actually transferred from the bacterium into the host where it gets integrated into the plant cell chromosome (i.e. host genome). Thus, crown gall disease is a naturally evolved genetic engineering process.
    [Show full text]
  • Vira and Virg Activate the Ti Plasmid Repabc Operon, Elevating Plasmid Copy Number in Response to Wound-Released Chemical Signals
    VirA and VirG activate the Ti plasmid repABC operon, elevating plasmid copy number in response to wound-released chemical signals Hongbaek Cho and Stephen C. Winans* Department of Microbiology, Cornell University, Ithaca, NY 14853 Edited by Patricia C. Zambryski, University of California, Berkeley, CA, and approved August 10, 2005 (received for review April 27, 2005) The vir genes of Agrobacterium tumefaciens tumor-inducing (Ti) be described. It is also quite possible that VirG-regulated genes may plasmids direct the transfer of oncogenic portion of the Ti (tumor- exist on replicons other than the Ti plasmid (9). inducing) plasmid that is transferred to plant cells (T-DNA) into Ti plasmids are generally referred to by the opines they can plant cells and are coordinately induced by plant-released phenolic catabolize, and the two best studied are often referred to as the chemical signals. We have used DNA microarrays, representing all nopaline-type plasmid originally found in strain C58 and the genes of the octopine- and nopaline-type Ti plasmids, to identify octopine-type Ti plasmid, which is found in virtually identical forms all Ti-plasmid-encoded genes in the vir regulons of both plasmids. in strains such as A6, B6, 15955, Ach5, and R10. All known Ti Acetosyringone (AS) induced the expression of all known members plasmids contain (i) one or more T-DNAs, (ii)thevir genes, which of the vir regulons, as well as a small number of additional genes. direct the transfer of T-DNA into plant cells, (iii) a set of tra and Unexpectedly, AS also caused a modest induction of virtually every trb genes, which direct the conjugation of the Ti plasmid, (iv) a large Ti plasmid gene.
    [Show full text]
  • Role of Plasmids in Plant-Bacteria Interactions
    Plasmids in Plant-Bacteria Interactions Schierstaedt et al. Curr. Issues Mol. Biol. (2019) 30: 17-38. caister.com/cimb Role of Plasmids in Plant-Bacteria Interactions Jasper Schierstaedt1, Nina Bziuk2, Nemanja bacteria interactions. Furthermore, we discuss tools Kuzmanović2, Khald Blau2, Kornelia Smalla2 and available to study the plant-associated mobilome, its Sven Jechalke3* transferability, and its bacterial hosts. 1Leibniz Institute of Vegetable and Ornamental Introduction Crops (IGZ), Department Plant-microbe systems, Plant-associated microorganisms are considered to Theodor-Echtermeyer-Weg 1, 14979 Großbeeren, be of great importance for plant health, plant Germany productivity and ecosystem functioning. They 2Julius Kühn-Institut - Federal Research Centre for expand the metabolic repertoire of plants, increase Cultivated Plants (JKI), Institute for Epidemiology the resource uptake and provide novel nutritional and Pathogen Diagnostics, Messeweg 11-12, 38104 and defense pathways (Berendsen et al., 2012; Braunschweig, Germany Berg et al., 2014). Therefore, the genetic 3Justus Liebig University Giessen, Institute for information provided by the plant microbiome is also Phytopathology, Heinrich-Buff-Ring 26-32, 35392 called the second genome of the plant (Berendsen Gießen, Germany et al., 2012). In the phytosphere, mutualistic associations were studied in great detail for rhizobia *[email protected] and mycorrhizae, rhizobacteria with plant growth promoting or biocontrol activity. However, also DOI: https://dx.doi.org/10.21775/cimb.030.017 parasitic interactions with plant pathogens are well- studied today. Plants are able to influence soil Abstract properties, e.g. by the release of nutrients and Plants are colonized by diverse microorganisms, secondary metabolites via root exudation, which are which may positively or negatively influence the used to combat pathogenic microorganisms while plant fitness.
    [Show full text]
  • Plant Binary Vectors of Ti Plasmid in Agrobacterium Tumefaciens with a Broad Host-Range Replicon of Prk2, Pri, Psa Or Pvs1
    American Journal of Plant Sciences, 2013, 4, 932-939 http://dx.doi.org/10.4236/ajps.2013.44115 Published Online April 2013 (http://www.scirp.org/journal/ajps) Review: Plant Binary Vectors of Ti Plasmid in Agrobacterium tumefaciens with a Broad Host-Range Replicon of pRK2, pRi, pSa or pVS1 Norimoto Murai Department of Plant Pathology and Crop Physiology, Louisiana State University and LSU AgCenter, Baton Rouge, USA. Email: [email protected] Received March 7th, 2013; revised April 4th, 2013; accepted April 13th, 2013 Copyright © 2013 Norimoto Murai. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. ABSTRACT This review chronicles the development of the plant binary vectors of Ti plasmid in Agrobacterium tumefaciens during the last 30 years. A binary vector strategy was designed in 1983 to separate the T-DNA region in a small plasmid from the virulence genes in avirulent T-DNA-less Ti plasmid. The small plant vectors with the T-DNA region have been simply now called binary Ti vectors. A binary Ti vector consist of a broad host-range replicon for propagation in A. tumeraciens, an antibiotic resistance gene for bacterial selection and the T-DNA region that would be transferred to the plant genome via the bacterial virulence machinery. The T-DNA region delimited by the right and left border sequences contains an antibiotic resistance gene for plant selection, reporter gene, and/or any genes of interest. The ColEI replicon was also added to the plasmid backbone to enhance the propagation in Escherichia coli.
    [Show full text]