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Plant Virus DNA Replication Processes in Agrobacterium

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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. The observation that geminivirus DNA rep- mutant), and deletion or inversion of nucleotide sequence lication functions are supported by the bacterial cellular between these two positions (Fig. 1). machinery provides evidence for the theory that these circular Generation of mutants of the four complementary-sense single-stranded DNA viruses have evolved from prokaryotic ORFs (Fig. 1) were carried out by site-directed mutagenesis episomal replicons. using an Altered Sites kit (Promega). The Cl-mutant was created by changing the ATG initiation codon at nucleotide Geminiviruses are a group of plant viruses characterized by position 2615 to a TAG termination codon. A premature TAA twin-shaped particles that are transmitted in the wild by insects stop codon was inserted into the C2 ORF by addition of AA and cause major crop losses worldwide. Their genomes consist after the T nucleotide at position 1525 (Fig. 1A). The C3- of either one or two circular single-stranded (ss) DNA species mutant was produced by a G to A mutation at position 1418 of 2.5-3.0 kb that are thought to replicate via a double- to create a premature TAG stop codon. The C4-mutant was stranded (ds) intermediate within the plant cell by a rolling produced by disruption of the ATG initiation codon at 2457, circle replication (RCR) mechanism (1, 2) analogous to that without affecting the coding of the overlapping Cl gene, as found in a class of eubacterial plasmids (3) and in some described in Rigden et al. (9). bacteriophages (4). The only viral ORF absolutely required for Mutants were initially produced as monomers and then replication, the geminiviral replication-associated protein cloned as head to tail repeats of 1.1, 1.5, or 2 units, containing (Rep or C1), has been shown by computer-assisted analysis of either one or two origins of replication (Fig. 1B), into the all geminiviruses characterized to date to contain three se- binary transformation vector pBinl9 (11). TLCV dimers (2.0 quence motifs common to RCR initiator proteins of the same mer) were constructed by ligation of an XbaI (wild type) or eubacterial plasmids and bacteriophages mentioned above BamHI dimer (Cl-mutant, not shown in Fig. 1) into the (5, 6). However, experimental evidence supporting an evolu- complementary cloning sites of pBinl9. VI and V2 mutant tionary origin for these DNA viruses as mobilized prokaryotic (Fig. 1A), 1.5-mer, and wild-type 1.5-mer constructs were episomes has so far been lacking. a monomer the Most geminiviruses are not mechanically transmissible and produced by cloning KpnI (containing specific are routinely inoculated into host plants in the laboratory by mutations/deletions) into the KpnI site of a pBinl9 clone a process that utilizes the ability of the bacterium Agrobacte- containing a 1246-bp KpnI/SalI fragment of wild-type TLCV, rium tumefaciens to transfer cloned tandem repeats of the viral thereby ensuring that the constructs contained only a single DNA into host cells (agroinoculation; ref. 7). Following agroin- copy of the mutated ORF. In the case of the Cl-mutant oculation, circular, monomeric DNA forms are generated 1.5-mer, the mutant KpnI monomer was cloned into a pBinl9 from the tandem repeats by recombination or a replicational clone containing a 1246-bp KpnI/SalI fragment obtained from release mechanism (1). During the course of our work on the the Cl-mutant TLCV monomer. Wild-type and complemen- mutational analysis of various ORFs in the geminivirus tomato tary-sense ORF mutant 1.1-mer constructs were produced by leaf curl virus (TLCV; refs. 8 and 9), we observed that DNA cloning a BamHI monomer (containing the specific muta- preparations taken from strains of A. tumefaciens that con- tions) into the BamHI site of a pBinl9 clone containing a tained cloned tandem repeats of the viral genome in the vector 276-bp BamHI/HindIII fragment of TLCV DNA. The HindIlI pBinl9 also contained monomeric, circular forms of the viral site is not present in the wild-type TLCV DNA and was DNA. In the present study we examine the accumulation of introduced by site-directed mutagenesis. these TLCV-specific DNA species in A. tumefaciens and demonstrate that the bacterial replication machinery, in asso- Abbreviations: TLCV, tomato leaf curl virus; RCR, rolling circle ciation with the viral Rep protein, provides functions to allow replication; RF, replicative form; Rep, replication-associated protein; ss, single stranded; ds, double stranded. *Present address: R. W. Johnson Pharmaceutical Research Institute, The publication costs of this article were defrayed in part by page charge P.O. Box 3331, Sydney 2001, Australia. payment. This article must therefore be hereby marked "advertisement" in tTo whom reprint requests should be addressed. e-mail: ali.rezaian@ accordance with 18 U.S.C. §1734 solely to indicate this fact. adl.hort.csiro.au. 10280 Downloaded by guest on September 30, 2021 Evolution: Rigden et al. Proc. Natl. Acad. Sci. USA 93 (1996) 10281 A 1/2766 B * on I ori 2 x B Bg KX B Bg K X A1DIII g lI' I 2.0 mer - Cl S 2 03 * _4-4.!23 - 4 -e- 4-,I -70 4-42-I -V-I 4- on I on 2 K X 9B K 2. LL . X_____) 1.5 mer S S on I on 2 H(;) B K X (;)B I oi 1.1 mer S S on B K X (; B Bg K 4. H i 1.5 mer S - S (single ori ) FIG. 1. Genome organization of TLCV and structures of linear tandem repeats used to produce pBinl9-based constructs. (A) ORFs shown by thick arrows are on both the virion-sense (encapsidated strand) of the viral DNA (VI and V2) and on the complementary-sense strand of the dsRF (Cl, C2, C3, and C4). Small arrows pointing to the ORFs indicate the positions of introduced mutations (nucleotide positions shown in brackets). IR is the intergenic region containing the origin of replication. The open box (o) just upstream of the stem-loop motif ( ) represents a region containing three iterative elements proposed as the binding site of the Rep (Cl) protein (10). B, BamHI; Bg, BglII; K, KpnI; S, SalI; X, XbaI. A HindIII site (H*) was introduced by site-directed mutagenesis at position 2642 to enable construction of the 1.1 mer. (B) Linear tandem repeats of TLCV DNA cloned into pBin19. Note that constructs 1, 2, and 3 each contain two ori but that the 1.1-mer (no. 3) contains only two of the three iterative elements within the Rep binding region of ori 1 ( ). There is only a single ori in construct 4. Constructs were introduced into A. tumefaciens strains C58 DNA probe (Fig. 3B) and (ii) by blot hybridization without and C58C1 and Escherichia coli strains DH5a and JM 101 by denaturation where it was the only TLCV DNA form that electroporation with a Gene Pulser apparatus (Bio-Rad) ac- hybridized to the TLCV probe (Fig. 3C). cording to the manufacturer's specifications. TLCV-specific supercoiled dsDNA as shown in Fig. 2 was Extraction and Analysis of DNA. Total nucleic acids were purified from A. tumefaciens harboring pBinl9 containing a extracted from TLCV-infected tomato plants (Lycopersicon 1.5-mer tandem repeat of the viral DNA and found to give esculentum Mill cv. Rutgers) as described (12). DNA was restriction digest patterns identical to those of wild-type TLCV extracted from bacteria using the miniprep method of Holmes dsDNA with five restriction enzymes (data not shown). Fur- and Quigley (13). Circular, RF viral DNA species were isolated thermore, agroinoculation of host plants with a tandem dimer from DNA preparations of A. tumefaciens by electrophoresis of this dsDNA purified from Agrobacterium produced infec- through 1.2% agarose Tris/acetate/EDTA gels and the ex- tions in host plants identical to those previously described for cised bands were purified using Geneclean (Bio 101) according wild-type virus (12). to the manufacturers specifications. Southern blot hybridiza- Products of Ti Plasmid Vir Genes Are Not Involved in TLCV tion was performed using 1.2% agarose gels in Tris/borate/ DNA Generation in Agrobacterium.
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