Walt Ream

TraG from RP4 and TraG and VirD4 from Ti Plasmids Confer Relaxosome Specificity to the Conjugal Transfer System of pTiC58

Plasmid conjugation systems are composed of two components, the DNA transfer and replication system, or Dtr, and the mating pair formation system, or Mpf. During conjugal transfer an essential factor, called the coupling protein, is thought to interface the Dtr, in the form of the relaxosome, with the Mpf, in the form of the mating bridge. These proteins, such as TraG from the IncP1 plasmid RP4 (TraG(RP4)) and TraG and VirD4 from the conjugal transfer and T-DNA transfer systems of Ti plasmids, are believed to dictate specificity of the interactions that can occur between different Dtr and Mpf components. The Ti plasmids of Agrobacterium tumefaciens do not mobilize vectors containing the oriT of RP4, but these IncP1 plasmid derivatives lack the trans-acting Dtr functions and TraG(RP4). A. tumefaciens donors transferred a chimeric plasmid that contains the oriT and Dtr genes of RP4 and the Mpf genes of pTiC58, indicating that the Ti plasmid mating bridge can interact with the RP4 relaxosome. However, the Ti plasmid did not mobilize transfer from an IncQ relaxosome. The Ti plasmid did mobilize such plasmids if TraG(RP4) was expressed in the donors. Mutations in traG(RP4) with defined effects on the RP4 transfer system exhibited similar phenotypes for Ti plasmid-mediated mobilization of the IncQ vector. When provided with VirD4, the tra system of pTiC58 mobilized plasmids from the IncQ relaxosome. However, neither TraG(RP4) nor VirD4 restored transfer to a traG mutant of the Ti plasmid. VirD4 also failed to complement a traG(RP4) mutant for transfer from the RP4 relaxosome or for RP4-mediated mobilization from the IncQ relaxosome. TraG(RP4)-mediated mobilization of the IncQ plasmid by pTiC58 did not inhibit Ti plasmid transfer, suggesting that the relaxosomes of the two plasmids do not compete for the same mating bridge. We conclude that TraG(RP4) and VirD4 couples the IncQ but not the Ti plasmid relaxosome to the Ti plasmid mating bridge. However, VirD4 cannot couple the IncP1 or the IncQ relaxosome to the RP4 mating bridge. These results support a model in which the coupling proteins specify the interactions between Dtr and Mpf components of mating systems.

Agrobacterium rhizogenes GALLS Protein Substitutes for Agrobacterium tumefaciens Single-Stranded DNA-Binding Protein VirE2

Agrobacterium tumefaciens and Agrobacterium rhizogenes transfer plasmid-encoded genes and virulence (Vir) proteins into plant cells. The transferred DNA (T-DNA) is stably inherited and expressed in plant cells, causing crown gall or hairy root disease. DNA transfer from A. tumefaciens into plant cells resembles plasmid conjugation; single-stranded DNA (ssDNA) is exported from the bacteria via a type IV secretion system comprised of VirB1 through VirB11 and VirD4. Bacteria also secrete certain Vir proteins into plant cells via this pore. One of these, VirE2, is an ssDNA-binding protein crucial for efficient T-DNA transfer and integration. VirE2 binds incoming ssT-DNA and helps target it into the nucleus. Some strains of A. rhizogenes lack VirE2, but they still transfer T-DNA efficiently. We isolated a novel gene from A. rhizogenes that restored pathogenicity to virE2 mutant A. tumefaciens. The GALLS gene was essential for pathogenicity of A. rhizogenes. Unlike VirE2, GALLS contains a nucleoside triphosphate binding motif similar to one in TraA, a strand transferase conjugation protein. Despite their lack of similarity, GALLS substituted for VirE2.

Crown-gall-resistant transgenic apple trees that silence Agrobacterium tumefaciens oncogenes

Crown gall disease is an economically significant problem in fruit and nut orchards, vineyards, and nurseries worldwide. Tumors on stems and leaves result from excessive production of the phytohormones auxin and cytokinin in plant cells genetically transformed by Agrobacterium tumefaciens. High phytohormone levels result from expression of three oncogenes transferred stably into the plant genome from A. tumefaciens: iaaM, iaaH, and ipt. The iaaM and iaaH oncogenes direct auxin biosynthesis, and the ipt oncogene causes cytokinin production. In contrast to other tissues, roots do not respond to high cytokinin levels, and auxin overproduction is sufficient to cause tumor growth on roots. Inactivation of iaaM abolished gall formation on apple tree roots. Transgenes designed to express double-stranded RNA from iaaM and ipt sequences prevented crown gall disease on roots of transgenic apple trees.

Agrobacterium rhizogenes GALLS Protein Contains Domains for ATP Binding, Nuclear Localization, and Type IV Secretion

Agrobacterium tumefaciens and Agrobacterium rhizogenes are closely related plant pathogens that cause different diseases, crown gall and hairy root. Both diseases result from transfer, integration, and expression of plasmid-encoded bacterial genes located on the transferred DNA (T-DNA) in the plant genome. Bacterial virulence (Vir) proteins necessary for infection are also translocated into plant cells. Transfer of single-stranded DNA (ssDNA) and Vir proteins requires a type IV secretion system, a protein complex spanning the bacterial envelope. A. tumefaciens translocates the ssDNA-binding protein VirE2 into plant cells, where it binds single-stranded T-DNA and helps target it to the nucleus. Although some strains of A. rhizogenes lack VirE2, they are pathogenic and transfer T-DNA efficiently. Instead, these bacteria express the GALLS protein, which is essential for their virulence. The GALLS protein can complement an A. tumefaciens virE2 mutant for tumor formation, indicating that GALLS can substitute for VirE2. Unlike VirE2, GALLS contains ATP-binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. Both GALLS and VirE2 contain nuclear localization sequences and a C-terminal type IV secretion signal. Here we show that mutations in any of these domains abolished the ability of GALLS to substitute for VirE2.

Agrobacterium tumefaciens and A. rhizogenes use different proteins to transport bacterial DNA into the plant cell nucleus

Agrobacterium tumefaciens and A. rhizogenes transport single‐stranded DNA (ssDNA; T‐strands) and virulence proteins into plant cells through a type IV secretion system. DNA transfer initiates when VirD2 nicks border sequences in the tumour‐inducing plasmid, attaches to the 5′ end, and pilots T‐strands into plant cells. Agrobacterium tumefaciens translocates ssDNA‐binding protein VirE2 into plant cells where it targets T‐strands into the nucleus. Some A. rhizogenes strains lack VirE2 but transfer T‐strands efficiently due to the GALLS gene, which complements an A. tumefaciens virE2 mutant. VirE2 and full‐length GALLS (GALLS‐FL) contain nuclear localization sequences that target these proteins to the plant cell nucleus. VirE2 binds cooperatively to T‐strands allowing it to move ssDNA without ATP hydrolysis. Unlike VirE2, GALLS‐FL contains ATP‐binding and helicase motifs similar to those in TraA, a strand transferase involved in conjugation. VirE2 may accumulate in the nucleus and pull T‐strands into the nucleus using the force generated by cooperative DNA binding. GALLS‐FL accumulates inside the nucleus where its predicted ATP‐dependent strand transferase may pull T‐strands into the nucleus. These different mechanisms for nuclear import of T‐strands may affect the efficiency and quality of transgenic events in plant biotechnology applications.

Import of Agrobacterium tumefaciens Virulence Proteins and Transferred DNA into Plant Cell Nuclei

Agrobacterium tumefaciens causes crown gall tumors on many dicotyledonous plant species when the bacteria infect wounded tissue (DeCleene and DeLey, 1976). This bacterium harbors a tumor-inducing (Ti) plasmid, where genes essential for tumorigenesis are located (Watson et al., 1975; Van Larebeke et al., 1974). The transferred DNA (T-DNA) portion of the Ti plasmid enters plant cells and integrates into nuclear DNA (Chilton et al., 1977, 1980; Willmitzer et al., 1980). Tumorous growth results from expression of three T-DNA genes that encode biosynthetic enzymes for two plant growth hormones, auxin (indole acetic acid) and cytokinin (isopentenyl adenosine) for reviews see Ream, 1989; Winans, 1992; Zambryski, 1992). There are many excellent reviews of Agrobacterium tumefaciens biology (Zambryski, 1992; Winans, 1992; Hooykaas and Beijersbergen, 1994; Citovsky, and Zambryski, 1993; Dreiseikelmann, 1994; Ream, 1989; Binns and Thomashow, 1988), and the most recent presents an outstanding treatment of nuclear localization of T-DNA (Sheng and Citovsky, 1996), the topic of this chapter.

VirE1-Mediated Resistance to Crown Gall in Transgenic Arabidopsis thaliana

ABSTRACT Crown gall disease, caused by Agrobacterium tumefaciens, remains a serious agricultural problem despite current biocontrol methods. Agrobacterium tumefaciens transfers single-stranded DNA (T-strands) into plant cells along with several virulence proteins, including a single-stranded DNA-binding protein (VirE2). In plant cells, T-strands are protected from nucleases and targeted to the nucleus by VirE2, which is essential for efficient transmission (transfer and integration) of T-strands. VirE1 is the secretory chaperone for VirE2; it prevents VirE2 from forming aggregates and from binding the T-strands in bacterial cells. Therefore, we hypothesized that sufficient quantities of VirE1 expressed in plant cells might block T-DNA transmission by preventing VirE2 from binding T-strands. Here we show that root explants from Arabidopsis thaliana plants that expressed virE1 formed 3.5-fold fewer tumors than roots from plants without virE1. Also, this resistance was specific for VirE2-mediated Agrobacterium transformation. Plants that have been genetically altered to resist crown gall may prove more effective than biological control.

Oligonucleotide-Directed Mutagenesis

Oligonucleotide-directed mutagenesis allows altering DNA in a specific location by hybridizing a complementary oligonucleotide primer containing the desired mutation to a circular single-stranded vector containing the target sequence. DNA polymerase extends the primer in vitro ; DNA ligase covalently joins the ends of the newly extended strand. This heteroduplex DNA now contains the mutation in its newly synthesized strand and the original sequence in the template strand. On transformation into E. coli, DNA repair and replication resolve this mismatch to produce colonies with either mutant or wild-type target DNA. Several methods can increase the chances of recovering mutant-containing clones. This chapter also illustrates how 58 bp can be deleted from virD2 and replaced with a 7-bp sequence that includes an NruI site.

Draft Genome Sequence of Erwinia billingiae OSU19-1, Isolated from a Pear Tree Canker

ABSTRACT Plant-associated Erwinia include pathogenic and nonpathogenic species. We report the 5.6-Mb genome sequence of Erwinia billingiae OSU19-1, isolated from a canker on a pear tree inoculated with Erwinia amylovora. OSU19-1 and a closely related European isolate, E. billingiae Eb661T, share many similarities including 40 kb of plasmid sequence.

Genetically engineered plants: greener than you think

Healthy, productive farmland separates humans from famine, making wise agricultural practices crucial to everyone. Plant scientists at universities and companies throughout the world endeavour to improve the quality, quantity, safety and sustainability of agricultural production, and their efforts have proven highly successful. Despite the remarkable success of a number of genetically engineered crop plants that have been grown and consumed – without incident – in large quantities since 1996, this technology faces increasing opposition in the United States and Europe. This article will discuss the major crops currently cultivated and provide information necessary to make informed decisions about plant biotechnology. Each new genetically engineered plant variety raises unique issues that must be considered on a case‐by‐case basis. Rejection or acceptance of all genetically engineered plants is unreasonable and will likely harm both the environment and the human condition.