Transfer of T-DNA From Agrobacterium to the Plant Cell

8/3/2019 Transfer of T-DNA From Agrobacterium to the Plant Cell

1/7

Plant Physiol. (1995) 107: 1041-1 047

Transfer of T-DNA from Agrobacterium to the Plant Cell'John R. Zupan and Patricia Zambryski*

Department of Plant Biology, 111 Koshland Hall, University of California, Berkeley, California 94720-31 02

Agrobucferium tumefuciens is the causative agent of crowngall, a disease of dicotyledonous plants characterized by atumorous phenotype. Earlier in this century, scientific in-terest in A. tumefaciens was based on the possibility that thestudy of plant tumors might reveal mechanisms that werealso operating in animal neoplasia. In the recent past, thetumorous growth was shown to result from the expressionof genes coded for by a DNA segment of bacterial originthat was transferred and became stably integrated into theplant genome. This initial molecular characterization of theinfection process suggested that Agrobacterium might beused to deliver genetic material into plants. The potentialto genetically engineer plants generated renewed interestin the study of A. fumefaciens. In this review, we concen-trate on the most recent advances in the study of Agrobuc-ferium-mediated gene transfer, its relationship to conjuga-tion, DNA processing and transport, and nuclear targeting.In the following discussion, references for earlier work canbe found in more comprehensive reviews (Hooykaas andSchilperoort, 1992; Zambryski, 1992; Hooykaas and Beijers-bergen, 1994).

OVERVIEW OF AGROBACTERIUM-MEDIATED CENETRANSFERA. tumefuciens is exploited by many plant biologists in

molecular and genetic studies to introduce DNA intoplants. Although best known for this practical application,the transfer of DNA from bacterium to plant comprisesfundamental biological processes, many of which arelargely uncharacterized.

During infection by A. tumefaciens, a piece of DNA istransferred from the bacterium to the plant cell (Fig. 1).This piece of DNA is a copy of a segment called the T-DNA(iransferred DNA). It is carried on a specific plasmid, theTi-plasmid (iumor-inducing), which is found in only asmall percentage of natural soil populations of A. tumefu-ciens. The T-DNA is delimited by 25-bp direct repeats thatflank the T-DNA. These borders are the only cis elementsnecessary to direct T-DNA processing. Any DNA betweenthese borders will be transferred to a plant cell. In contrastto other mobile DNA elements, T-DNA does not itselfencode the products that mediate its transfer. Wild-type

Research on Agrobucterium is funded by the National Science* Corresponding author; e-mail zambryski8mendel.Foundation (DCB 8915613).

berkeley.edu; fax 1-510-642-4995.

T-DNAs encode enzymes for the synthesis of the plantgrowth regulators auxin and cytokinin and the productionof these compounds in transformed plant cells results inthe tumorous phenotype. In addition, wild-type T-DNAalso encodes enzymes for the synthesis of nove1 amino acidderivatives called opines. The Ti-plasmid encodes enzymesfor their catabolism; hence, Agrobucferium has evolved togenetically commandeer plant cells and use them to pro-duce compounds that they uniquely can utilize as a car-bon/nitrogen source. Additional profit derives from theability of opines to stimulate conjugation of the Ti-plasmid,thereby increasing the bacterial population that can utilizeopines (reviewed by Greene and Zambryski, 1993).

The processing and transfer of T-DNA are mediated byproducts encoded by the vir (virulence) region, which isalso resident on the Ti-plasmid (Stachel and Nester, 1986).Those vir genes, whose products are directly involved inT-DNA processing and transfer, are tightly regulated sothat expression occurs only in the presence of woundedplant cells, the targets of infection. Control of gene expres-sion is mediated by the VirA and VirG proteins, a two-component regulatory system (reviewed by Winans, 1992).VirA detects the small phenolic compounds released bywounded plants resulting in autophosphorylation (Fig. 1,step 1). VirA phosphorylation of VirG then leads to acti-vation of vir gene transcription.

Following vir gene induction, the production of a trans-fer intermediate begins with the generation of the T-strand,an ss copy of the T-DNA (Stachel et al., 1986). VirDl andVirD2 are essential for this process (Filichkin and Gelvin,1993). Together, VirDl /D2 recognize the 25-bp border se-quence and produce an ss endonucleolytic cleavage in thebottom strand of each border (Fig. 1, step 2) These nicks areused as the initiation and termination sites for T-strandproduction. After nicking, VirDZ remains tightly associatedwith the 5' end of the T-strand. The lone VirD2 at the5' endgives the T-complex a polar character that may ensure that,in subsequent steps, the 5' end is the leading end. T-strandproduction is thought to result from the displacement ofthe bottom strand of the T-DNA between the nicks. Recentwork has suggested that the early steps in T-strand pro-duction are evolutionarily related to other bacterial sys-tems that produce ssDNA, such as during conjugation(Table I; discussed below).

Abbreviations: ds, double-stranded; NLS, nuclear localizationsignal; ss, single-stranded.

1041


2/7

1042 Zupan and Zambryski Plant Physiol. Vol. 107, 1995

44

@Induction dvir p n e wrpression-Roductim dT-strnd byVirD#DZ- CYTOPLASM 1cytoplpsmicReceptas

PLANTCELLFigure 1. Basic steps in the transformation of plant cells by A. tumefaciens. Each step is described i n the text.

The T-strand must trave1 through numerous membranesand cellular spaces before arrival in the plant nucleus.Thus, to preserve its integrity, it was hypothesized that theT-strand likely travels as an ssDNA-protein complex. VirE2is an inducible ss nucleic acid-binding protein encoded bythe virE locus that binds without sequence specificity.VirE2 binds tightly and cooperatively, which means that aT-strand would be completely coated (Fig.1, step 3). Con-sequently, degradation by nucleases would be preventedand, indeed, in vitro binding of VirE2 renders ssDNAresistant to nucleolytic degradation. Finally, binding ofVirE2 unfolds and extends ssDNA to a narrow diameter of2 nm, which may facilitate transfer through membranechannels. The T-strand along with VirD2 and VirE2 aretermed the T-complex.

Subsequently, the T-complex must exit the bacterial cell(Fig. 1, step 4 ) , passing through the inner and outer mem-branes as well as the bacterial cell wall. It must then crossthe plant cell wall and membrane (Fig. 1, step 5) . Onceinside the plant cell, the T-complex targets to the plant cellnucleus and crosses the nuclear membrane (Fig.1, step 61,after which the T-strand becomes integrated into a plantchromosome (Fig.1, step 7). In the context of experimentalchronology and relevant results, steps 1 through 3 (Fig. 1)have been well studied. Current and recent research hasrelated to steps 4 and 6. Entering the plant cell (step 5 ) and

the mechanics of integration (step 7) are almost completelyuncharacterized.

RELATIONSHIP OF T-DNA TRANSFER 1'0CONJUGATIVE DNA TRANSFEREvidence supporting the hypothesis that Agrobacterium-

plant DNA transfer and bacterial conjugative DNA transferare evolutionarily related has been rapidly accu mulating.This hypothesis was first proposed following the discoveryof the T-strand (Stachel and Zambryski, 1986; Stachel et al.,1986) and was based on striking parallels with the synthe-sis and transfer of conjugal DNA. First, short sequencesrequired in cis (oriT and T-DNA right border) are function-ally polar in directing DNA transfer. Transfer i:; initiatedby nicks in these sequences, and reversing their orientationinhibits DNA transfer. Second,ss linear DNA is transferredfollowing its displacement from the plasmid. Fin,illy, DNAtransfer will occur only if the donor and recipient cell are inintimate contact.

Recent studies have not only confirmed these functionalanalogies but extended them to include similarikies in theamino acid sequences, gene organization, and physicalproperties of the trans-acting enzymes involved. Functionaland molecular similarities of T-DNA transfer antl conjuga-tion are most completely characterized with respect to the


3/7

Transfer of T-DNA from Agrobacterium to the Plant Cell 1043

Table 1. Comparison of the similarities between T-DNA transfer and bacterial conjugative DNAtransfer in the RP4 systemsimilarity are underlined.

Key elements of each system are in bold and elements with nucleotide or amino acid sequence

Characteristic Proposed Analogy between RP 4 and T-DNA Transferlnitiation siteEndonuclease assemblyNicking

DNA transfer initiates at the right border (T-DNA) and at thenick region of oriT (RP4); see text for sequence similarity.V i r D 1 , E form a complex on the right border (T-DNA) and

Tra!,J along with TraH assemble at the nick site (RP4).Site-specific DNA cleavage by V i r g (T-DNA) and Trai (RP4),

both of which remain attached to the 5 end of the DNA tobe transferred.

T-strand, ssT-DNA copy, is released from Ti-plasmid by anunknown mechanism followed by the cooperative bindingof VirE2, an ssDNA-binding protein, to form T-complex.Transferred ssDNA i s unwound from RP4; no conjugation-specific, ssDNA-binding protein has been identified.

Transfer T-DNA transfer and ss conjugative DNA transfer (RP4) occursdirectionally from 5 to 3 .

Export Products of Vir! and operons primarily form structure forexport of DNA from donor and may have a role in import oftransferred DNA into recipient.

Transfer intermediate

plasmid RP4, a broad-host-range plasmid originally iso-lated from a strain of Pseudomonas (Table I). The study ofRP4 and T-DNA transfer has been mutually informative(reviewed by Lessl and Lanka, 1994). First, the develop-ment of in vitro techniques for the study of DNA process-ing in the RP4 system has led directly to the biochemicalcharacterization of some Vir proteins. Second, comparativeanalysis of the RP4 Tra2 operon, involved in transfer ofDNA from donor to recipient, and the Agvobacterium virBoperon has suggested possible roles for some of the pro-teins encoded by these operons in DNA transfer.

Processing of the Transfer lntermediate in Agrobacteriumand RP4

DNA transfer begins with an ss nick at specific sites inboth the T-DNA and the conjugative DNA in RP4. In vivo,TraI and TraJ cooperate as the RP4 relaxase to nick oviT. InAgrobacterium, VirDl /D2 cleave the T-DNA border se-quences between the third and fourth bases. Both nick sitescontain a 12-nucleotide consensus sequence (AC/TC/AT/CATCCTGC/TC/A) that has also been found among var-ious rolling circle-type replicons (Waters and Guiney, 1993;Lessl and Lanka, 1994). The oriT of RSF1010, the IncQconjugative plasmid, and its cognate relaxase (mobiliza-tion) proteins actually can substitute for T-DNA borders inthe transfer of DNA to a plant cell.

In vitro, TraI and VirD2 are each sufficient, in the pres-ente of Mg2+,to produce nicks in ss oligonucleotides bear-ing their respective cognate nick sites (Pansegrau et al.,1993; refs. in Lessl and Lanka, 1994). In the presence of anexcess of cleavage products, both TraI and VirD2 can alsocatalyze the opposite reaction, joining two pieces ofssDNA. It is curious that VirD2 can catalyze the cleavage oforiT in an ss oligonucleotide but TraI cannot cleave a ssT-DNA right border. The ability to recognize other nick sitesis consistent with the hypothesis that VirD2 initiates the

integration of T-strand by joining it to the 3 side of a n ssnick in plant DNA. Consequently, if it also binds to a plantDNA site prior to joining, VirD2 may have evolved totolerate more variability in the DNA sequences to which itwill bind. If the cognate sites are present in supercoiled dsplasmids, TraJ and VirDl must be included with TraI andVirD2, respectively, to obtain nicking. Neither complexwill cleave its cognate nick site if it is present in the form ofa relaxed ds circle or a linear double strand.

At the molecular level, TraI has three highly conservedmotifs at the N terminus that it shares with VirD2 andrelaxases of other conjugative systems (Pansegrau et al.,1994). Motif I contains a Tyr at position 22 in TraI thatforms a phosphodiester between its aromatic hydroxylgroup and the 5 phosphoryl group of the DNA duringcleavage (Pansegrau et al., 1993). Motif I11 in TraI containstwo Hiss necessary for cleavage that may serve to activatethe Tyr and for coordinate binding of Mg2+.Recognitionof the binding site 3 to the nick may be the role of motif11. Critica1 amino acids for these functions have been iden-tified in TraI by mutagenesis and are predicted to be con-served in VirD2 based on sequence comparison. The Tyrto which the transferred ssDNA becomes attached occursat position 29 in VirD2. This amino acid is not only withina context equivalent to that around position 22 in TraIbut also cannot be altered without abolishing nickingactivity.

The role of VirDl may be deduced by comparison toTraJ, its presumed counterpart in the RP4 system. Previ-ously, bacterial extracts enriched in VirDl were suggestedto have topoisomerase I activity. This does not, however,appear to be an enzymatic activity of purified VirDl. InRP4, TraJ first binds to the 3 side of oriT and then recruitsTraI. This sequence of events appears to be specific to thenicking of supercoiled dsDNA. By analogy, VirDl may firstrecognize and bind the T-DNA border, which promotesVirD2 binding.


4/7


Export from BacteriumExport of the T-complex from Agrobucterium is one of the

steps in the transfer process about which very little isknown. The steps in generating a transferable T-DNA copywere linked to one or more of the vir operons. Export,however, was a function without an operon. Early workshowed that virB was necessary for tumorigenesis but wasnot required for T-strand or T-complex formation (Stacheland Nester, 1986). Sequencing of virB suggested that itsgene products may be involved in the transfer process(reviewed by Zambryski, 1992). The 11 open readingframes of the virB locus code for proteins that exhibit thepredicted features necessary for forming a membrane-as-sociated export apparatus, including hydrophobicity,membrane-spanning domains, and/or N-terminal signalsequences that target protein out of the cytoplasm.

Subcellular localization studies confirmed the membraneassQciationof seven VirB proteins (Thorstenson et al., 1993;Beijersbergen et al., 1994; Thorstenson and Zambryski,1994). A11 seven proteins, however, were distributed be-tween the inner and outer membranes, in proportions thatvaried depending on the fractionation method (Thorsten-son et al., 1993). It has been suggested that these VirBproteins are so tightly juxtaposed in a complex that spansboth membranes that they fractionate randomly betweenthe inner and outer membranes.

By sequence comparison, virB has been shown to beevolutionarily related to other bacterial gene clusterswhose products form a membrane-associated complex in-volved in fimbriae formation, protein secretion, or DNAuptake (reviewed by Lessl and Lanka, 1994). Comparisonof a genetic element of RP4, the Tra2 region, to virB hasshed light on the process of DNA transfer in these twosystems. The Tra2 region of RP4 encodes 11 proteins in-volyed in the DNA-transfer step of conjugation, probablyforming the pilus or pilus precursors. Six of the Tra2 pro-teins are highly similar to VirB proteins, especially in fea-tures that suggest membrane localization, e.g. hydrophobicdomains (Lessl et al., 1992). The genes for five of these Tra2proteins are collinear with their virB counterparts. At leasttwo of these proteins, Vir82 and TrbC, show some se-.quente similarity to TraA (from Escherichiu coli F factor), abona fide pilus subunit.

Two VirB proteins, VirB4 and 11, are candidates to pro-vide the energy for translocation of the T-complex. Bothlocalize to the inner membrane, consistent with this pro-posed function. VirB4 has a nucleotide-binding site that isessential for virulence (Berger and Christie, 1993).VirBll isreported to associate with the cytoplasmic face of the innermembrane, and it exhibits both ATPase and protein kinaseactivity. VirBll is related by amino acid sequnce to TrbBof the Tra2 operon, which possesses similar enzymaticactivities. Proteins similar to TrbB are found in a variety ofbacteria usually in membrane-associated, multi-proteincomplexes that function in import or export of protein andDNA. Lessl and Lanka (1994) suggested that TrbB-likeproteins may form a class of ATPases that function specif-ically in complexes that translocate macromolecules and

macromolecular complexes across the bacterial i nner andouter membranes.

The virB operon also appears to be closely relaled to thePtl operon (Weiss et al., 1993). The Ptl operon (Bordetellapertussis) encodes products responsible for expo *t of per-tussis toxin protein. Five of the encoded proteins are sig-nificantly similar, and four of the genes for these proteinsare collinear. Within TrbB-like proteins, VirBll m d TrbBform a closely related subgroup that also inclutles PtlH.Therefore, the machinery to export the pertussis toxin mayhave evolved from the same progenitor as the s-wtems totransfer DNA between bacteria and from bacterium toplant. Possibly the t ransport systems for T-DNA and con-jugation evolved from a protein export system and nowrecognize only the protein component of the transfer inter-mediates. The T-strand, essentially masked by VirD2 andVirE2, may be coincidentally carried along as tri nsport ismediated by protein-protein interactions (Thorstenson andZambryski, 1994).

Aside from a requirement for activated VirG i o inducetranscription, little is known about additional, more subtlemechanisms that may regulate virB expression. The virBoperon is transcribed as a single polycistronic message thatincludes a1111open reading frames, and most mutagenesisstrategies tend to be polar, which requires that the virBoperon be reconstructed to express genes downstream ofthe one being tested. Thus, assessing expression require-ments and the necessity of individual loci for viiulence isextremely labor intensive in virB. Recent, in-depth studies,however, have shown virB2 through virBll to be absolutelyrequired for virulence and virBl mutants severely attenu-ated in virulence (Dale et al., 1993; Berger and Christie,1994).Expression of severa1 virB genes depends on cis-actingelements. Mutants for these virB loci were not restored towild-type virulence unless the complementing phsmid in-cluded adjacent sequences or entire adjacent genes (Dale etal., 1993; Berger and Christie, 1994). These requirements foradjacent genes can be partially explained by the proposedtranslational coupling between virBl/2 and virB8 19. Accu-mulation of virB9 was shown to be much reduced in bothvirB7 and virB8 mutants (Berger and Christie, 1994). Byanalogy to P pilus formation, in which coordinate proteinsynthesis can stabilize individual proteins througli protein-protein interaction (Kuehn et al., 1993), the accumiilation ofsome VirB proteins may require other VirB proteins. Aninteraction with another protein(s) may promcite eitherstability or proper localization before degradatioli. In fact,both accumulation and localization of VirB3 are dependenton VirB4 (Jones et al., 1994). Agrobacterium thai did notexpress VirB4 accumulated greatly reduced cellular levelsof VirB3, which fractionated with the inner membrane. Inthe presence of VirB4, wild-type levels of VirB3 ippearedin both the inner and outer membrane fractions.

Almost nothing is known about the interaction(s) be-tween the putative VirB complex and T-complex presumedto occur prior to and during export. VirD4 has been sug-gested to mediate these interactions based on its require-ment for virulence and its inner membrane localization


5/7


(Thorstenson and Zambryski, 1994). VirD4 is not requiredfor T-strand or T-complex formation. TraG is the VirD4counterpart in the RP4 system. Because TraG is requiredfor conjugation but is involved in neither nick-site cleavageor pilus formation, it has been speculated to be involved inlinking the mobilized DNA to the pilus during transfer.The functional analogy between VirD4 and TraG is sup-ported by the demonstration that VirD4 and the VirB pro-teins can substitute for TraG and Tra2 products, respec-tively, in mediating the transfer of RSFlOlO betweenAgrobacteria.

A11 evidence to date supports the hypothesis that virB isinvolved in the transfer of the T-complex out of the bacte-rium. Sequence data and comparison to conjugative DNAtransfer systems indicate that at least some of the virBproducts form a membrane-associated complex that maybe functionally similar to a pilus. The remaining VirB pro-teins may assist in assembly of the complex or provideancillary functions during T-complex transfer (e.g. ATPaseactivity for energy).

SS INTERMEDIATEAlthough the early evidence for the T-strand intermedi-

ate was compelling, the debate about what is the "real"T-DNA copy has nevertheless continued for the last 10years. Evidence for ds circle as well as ds linear moleculeshas been presented (reviewed by Zambryski, 1992).Both ofthese ds forms, however, are found only in the bacteriumand probably result from the nicks in the 25-bp repeats,either promoting recombination to generate circles or pro-moting nonspecific opposite-strand breakage during DNAextraction. In two recent papers, however, the debate hashopefully been put to rest. Two very different strategieswere used to assay the nature of the T-DNA copy uponarrival in the plant cell. In both cases, the results moststrongly supported an ss transfer intermediate.

Yusibov et al. (1994) monitored (a) the synthesis anddisappearance of the T-DNA copy within Agrobacteriumand (b) the appearance of a PCR-detected T-DNA segmentin tobacco protoplasts. The disappearance of the T-DNAcopy from Agrobacterium was correlated with the appear-ance in the plant cytoplasmic fraction of the T-DNA seg-ment. The latter product was not amplified if the plantcytoplasmic fraction was first treated with S1 nuclease thatpossessed no detectable ds DNase activity. Control exper-iments excluded the possibility that lysed bacteria were thesource of the T-DNA template. The lack of a PCR productafter nuclease S1 treatment supports the hypothesis thatthe transfer intermediate is indeed an ss molecule.

In the second study, a sensitive extrachromosomal re-combination assay was used (Tinland et al., 1994). Briefly,the strategy was to design a T-DNA with a GUS genedivided into two overlapping segments. In planta recom-bination is then required to yield a full-length gene. Extra-chromosomal recombination that takes place early afterentry of the T-complex into the nucleus can discriminatebetween the transfer of dsDNA and ssDNA. If the transferintermediate is ds, recombination should produce an intactGUS regardless of whether the segments in the T-DNA are

of the same or opposite polarity. If the transfer intermedi-ate is ss, however, then a complete GUS can be obtainedthrough recombination only if the segments are of oppositepolarity. GUS activity in infected protoplasts was an orderof magnitude greater from the T-DNA bearing GUS seg-ments of opposite polarity relative to GUS segments of thesame polarity. Thus, this study and that by Yusibov etal. (1994) provide strong confirmation for an ss transferintermediate.

NUCLEAR IMPORT OF T-COMPLEXIntegration of T-strand requires nuclear uptake, which is

a tightly regulated process that can occur only through,thenuclear pore. Proteins larger than approximately 40 kDrequire an NLS that mediates their nuclear uptake. TheT-complex has three components that could potentiallycontribute the signal(s) for nuclear import. The T-stranditself is unlikely to possess a signal for nuclear import,since any DNA between borders will be transferred. Nu-clear transport of T-complex is most likely mediated by itsassociated proteins, VirD2 or VirE2.

Sequence analysis of VirD2 revealed that the N terminusis 85% conserved among strains of Agrobacterium; the Cterminus is only 25% conserved, but the highest similarityis in the last 30 amino acids (reviewed by Zambryski, 1992).Within this domain, a sequence homologous to the bipar-tite type of NLS was found (Howard et al., 1992). Thenuclear localizing function of this sequence was confirmedby fusing its coding region to the GUS reporter gene andtransiently expressing this construct in tobacco protoplasts(Howard et al., 1992). The nuclear localization of the fusionprotein was inferred from the accumulation of the blueGUS product in the nucleus. When the putative NLS wasdeleted from full-length VirD2 fused to GUS, the GUSproduct was only in the cytoplasm (Howard et al., 1992).Nuclear accumulation of transiently expressed VirD2 hasalso been demonstrated by immunolocalization (Tinland etal., 1992). That the C-terminal bipartite NLS sequence has arelevant biological function was confirmed by the observa-tion that Agrobacterium was severely reduced in tumorige-nicity when the two basic stretches of the bipartite NLSwere deleted from VirD2 (Shurvinton et al., 1992).

Although VirD2 possesses a functional NLS necessary tomediate nuclear uptake of T-complex, the T-complex is anextremely large nucleoprotein complex (reviewed by Zam-bryski, 1992). In some strains of Agrobacterium, the T-strandis 20 kb. This T-strand would bind approximately 600molecules of VirE2 and would have a calculated length of3600 nm, 60 times the thickness of the nuclear membrane.Including a single VirD2, the T-complex has a mass ofabout 50 X 106 D. 1s a single bipartite NLS capable ofmediating the nuclear uptake of a complex this large?

If additional NLSs are required for nuclear uptake, theymay be supplied by VirE2, the most abundant proteincomponent of the T-complex. Sequence analysis revealedtwo potential bipartite NLSs in VirE2. As with VirD2, thenuclear localization of VirE2 was tested by fusing its cod-ing regions to GUS. Although mutants in which one or theother of the NLSs were deleted exhibited some nuclear


6/7


transport, both NLSs were required for maximum accumu-lation in the nucleus (Citovsky et al., 1992). Thus, VirE2provides NLSs along the entire length of the T-complex.The multiple NLSs of VirE2 may be functionally importantfor uninterrupted nuclear import of T-complex, e.g. keep-ing the cytoplasmic and nucleoplasmic sides of the nuclearpore open simultaneously.The presence of NLSs in VirE2 raises the question ofwhether the NLS of VirD2 is superfluous or has a uniquerole in the import of T-complex. That Agrobacterium bearingan NLS-deleted virD2 was severely attenuated in virulencesuggests the latter. Possibly, VirD2 initiates uptake andensures that the 5 end of the T-strand enters the nucleusfirst. Leading with the 5 end may be a common feature ofthe translocation of ss nucleic acids across the nuclear pore.For example, export of the 75s rRNA (Balbioni Rings) fromthe nuclei of salivary gland cells in Chironomus tentansinitiates at the 5 end (Mehlin et al., 1992).

Compelling evidence for an in planta role for VirE2 isderived from experiments using wild type or virE2 mutantsof Agrobacterium in combination with transgenic tobaccoplants expressing wild-type or mutant VirE2. In the first setof experiments, avirulent virE mutants of Agrobacteriumwere restored to wild-type virulence when inoculated ontobacco expressing wild-type VirE2 (Citovsky et al., 1992).Complementation was reduced on plants expressing trans-genic VirE2 in which either NLS had been deleted(Citovsky et al., 1992). Potentially, two functions might becomplemented in planta, nuclear import or protection fromnucleases, because mutagenesis of either NLS also reducedss nucleic acid-binding activity in vitro. In the second set ofexperiments, wild-type VirE2-expressing transgenic to-bacco was partially resistant to infection by a virulent (i.e.VirE2+) strain of Agrobacterium (Citovsky et al., 1994). Re-sistance was maximal in plants expressing mutant VirE2that was unable to bind ssDNA but still contained bothNLSs. Thus, nuclear import is at least one of the in plantafunctions that might be inhibited by VirE2 expressed intransgenic plants (Citovsky et al., 1994).

The recalcitrance of monocots to transformation byAgrobacterium is a puzzle that remains unsolved. Althoughconsidered unlikely given the evolutionary conservation ofbipartite NLSs from plants to animals, nuclear localizationwas one of the steps at which a block could possibly occur.Both VirD2 and VirE2 in fusion to GUS, however, accumu-lated in the nuclei when transiently expressed in epidermalcells of maize leaves and immature sections of the root(Citovsky et al., 1994). Thus, both of the Agrobacteriumproteins associated with the T-complex are recognized bythe nuclear import machinery of a monocot cell, whichsuggests that the complex itself could be imported into thenucleus.

Clearly, the study of Agrobacterium has led to discoveriesin other areas of biology, from plant-bacterial signaling toconjugation to nuclear import. Most recently, the study ofnuclear import in the process of T-complex transfer unex-pectedly revealed a possible developmental component tothe regulation of nuclear import (Citovsky et al., 1994).Transiently expressed VirD2 and VirE2 GUS fusions accu-

mulated in the nuclei of leaf and immature root epidermalcells of both tobacco and maize. In mature root epidermis,however, both fusion proteins remained cytoplasmic. Itmay be that nuclear import of VirD2 and VirE2 is mediatedby NLS-binding proteins that are not produced in a11 celltypes. Developmentally specific gene expression could po-tentially be achieved by a cell type-specific comp iement ofNLS-binding proteins. Different subsets of trartscriptionfactors would be admitted to the nucleus, depending onwhich NLS-binding protein(s) were expressed ir that celltype. Increasingly, nuclear import regulation is beingshown to have a role in the control of transcript on factoractivity (Whiteside and Goodbourn, 1993). Fuither evi-dente of multiple pathways for nuclear import is indicatedby transgenic tobacco expressing VirE2. Nuclear importassociated with normal cellular processes is appal-ently notsignificantly inhibited by the transgenic protein :Citovskyet al., 1994).

Integration of foreign DNA into a host chrontosome isthe final step of the T-DNA transfer process (Fig.1,step 7)and it is this function, unique among plant pathoGens, thatis largely responsible for the intense investigation ofAgrobacterium. However, the mechanics of this stvp remainlargely unknown. The best model to date comparts T-DNAintegration to illegitimate recombination (Gheysen et al.,1991). It is suggested that the VirD2-bound 5 end of theT-strand joins a nick in plant DNA. The plant TINA mayfurther unwind to form a gap, and the 3 ertd of theT-strand may pair with another region of plant DNA closeby. Plant repair and recombination enzymes inay thenfunction to covalently join the 3 end to plant DNA. Thesereactions would result in the introduction of thc T-strandinto one strand of plant DNA. Torsional strain would thenresult in the introduction of a nick into the opposite strandof plant DNA. Gap repair and DNA synthesis using theT-strand as template result in the final integratiort product.

FUTURE DlRECTlONSA. tumefacierzs is the only known naturally occurring

organism capable of genetically transforming a plant cell. Itis both a tool for the plant molecular biologist and a reser-voir of fascinating biology. Although some of the processeswhereby DNA is transferred from Agrobacterium to a plantare understood in some detail, others are not. The relation-ship between virB products and T-complex export is stillunresolved. Although an export-specific channel is the pre-dicted virB product, nothing is known about the details ofits assembly, structure, or function. In conjuga ve DNAtransfer, the pilus, which in some bacteria is a proiuct of anoperon similar to virB, has a role in contacting the recipientcell. Whether any of the virB products has a role in (pilustype) recognition of the plant cell surface is not krtown. Thebinding of Agrobacterium to the plant cell surface LSanotherpoint in the infection process at which monocos may beresistant. Monocots are known to produce inducers of virgene expression and, as discussed above, are conipetent toimport T-complex into the nucleus. Thus, it mey be thatproteins on the surface of Agrobacterium are inable torecognize receptors on the surface of the mortocot cell,


7/7


thereby preventing attachment and transfer of T-DNA.Finally, nothing is known about the enzymology of inte-gration. No plant factors have been implicated in this pro-cess. VirD2 is presumed to catalyze the integration of T-strand into the plant genome; however, no data have beenobtained to support this hypothesis. It is also not knownwhether VirE2 plays a role in this process, possibly per-forming a function analogous to that of RecA (West, 1992).Future research should address these questions and may,as in the past, shed considerable light on other areas ofbiology.

ACKNOWLEDCMENTWe thank the past and present members of the Zambryski

laboratory for their support and helpful discussions.Received November 18, 1994; accepted January 18, 1994.Copyright Clearance Center: 0032-0889/95/l07/lO41/07.

LITERATURE ClTEDBeijersbergen A, Smith SJ, Hooykaas PJJ (1994) Localization andtopology of VirB proteins of Agrobacterium tumefuciens. PlasmidBerger BR, Christie PJ (1993) The Agrobucterium tumefuciens virB4gene product is an essential virulence protein requiring an intactnucleoside triphosphate-binding domain. J Bacteriol 175: 1723-1734Berger BR, Chris tie PJ (1994) Genetic complementation analysis ofthe Agrobucterium tumefuciens virB operon: virB2 through virBllare essential virulence genes. J Bacteriol 176 3646-3660Citovsky V, Wamick D, Zambryski P (1994) Nuclear impor t ofAgrobucterium VirD2 and VirE2 proteins in maize and tobacco.Proc Natl Acad Sci USA 91:3210-3214Citovsky V, Zupan J, Warnick D, Zambryski P (1992) Nuclearlocalization of Agrobucterium VirE2 protein in plant cells. ScienceDale EM, Binns AN, Ward JEJ (1993) Construction and character-ization of Tn5virB, a transposon that generates nonpolar muta-tions, and its use to define virB8 as an essential virulence gene inAgrobucterium tumefaciens. J Bacteriol 175 887-891Filichkin SA, Gelvin SB (1993) Formation of a putative relaxationintermediate during T-DNA processing directed by the Agrobucte-rium tumefuciens VirD1,DZ endonuclease. Mo1 Microbiol8: 915-926Gheysen G, Villarroel R, Van Montagu M (1991) Illegitimaterecombination in plants: a model for T-DNA integration. GenesDev 5 287-297Greene EA, Zambryski PC (1993) Agrobucteriu mate in opine dens.Curr Biol 3: 507-509Hooykaas PJ, Schilperoor t RA (1992) Agrobucterium and plantgenetic engineering. Plant Mo1 Biol 19: 15-38Hooykaas PJJ, Beijersbergen AGM (1994) The virulence system ofAgrobucterium tumefaciens. Annu Rev Phytopathol 3 2 157-179Howard EA, Zupan IR, Citovsky V, Zambryski PC (1992) TheVirD2 protein of A. tumefuciens contains a C-terminal biparti tenuclear localization signal: implications for nuclear uptake ofDNA in plant cells. Cell 68: 109-118Jones AL, Shir asu K, Kado CI (1994) The product of the virB4 geneof Agrobacterium tumefuciens promotes accumulation of VirB3protein. J Bacteriol 176:5255-5261Kuehn MJ, Ogg DJ, Kihlberg J, Slonim LN, Flemmer K, BergforsT, Hultgren SJ (1993) Structural basis of pilus subunit recogni-tion by the PapD chaperone. Science 262: 1234-1241

3 2 212-218

256 1802-1805

Lessl M, Balzer D, Pansegrau W, Lanka E (1992) Sequence simi-larities between the RP4 Tra2 and the Ti virB region stronglysupport the conjugation model for T-DNA transfer. J Biol Chem

Lessl M, Lanka E (1994) Common mechanisms in bacterial conju-gation and Ti-mediated T-DNA transfer to plant cells. Cell 77:Mehlin H, Daneholt B, Skoglund U (1992) Translocation of aspecific premessenger ribonucleoprotein partcle through the nu-clear pore studied with electron microscope tomography. CellPansegrau W, Schroder W, Lanka E (1993) Relaxase (TraI) of

IncPa plasmid RP4 catalyzes a site-specific cleaving-joining re-action of single-stranded DNA. Proc Natl Acad Sci USA 90:Pansegrau W, Schroder W, Lanka E (1994) Concerted action of

three distinct domains in the DNA cleaving-joining reactioncatalyzed by relaxase (TraI) of conjugative plasmid RP4. J BiolChem 269: 2782-2789

Shurvinton CE, Hodges L, Ream W (1992) A nuclear localizationsignal and the C-terminal omega sequence in the Agrobucteriumtumefuciens VirD2 endonuclease are important for tumor forma-tion. Proc Natl Acad Sci USA 89: 11837-11841

Stachel SE, Nester EW (1986) The genetic and transcriptionalorganization of the vir region of the A6 Ti plasmid of Agrobuc-terium tumefuciens. EMBO J 5: 1445-1454Stachel SE, Timmerman B, Zambryski P (1986) Generation ofsingle-stranded T-DNA molecules during the initial stages ofT-DNA transfer from Agrobucterium tumefaciens to plant cells.Nature 322: 706-712Stachel SE, Zambryski PC (1986) Agrobucterium tumefuciens andthe susceptible plant cell: a nove1 adaptation of extracellularrecognition and DNA conjugation. Cell 47: 155-157

Thorstenson YR, Kuldau GA, Zambryski PC (1993) Subcellularlocalization of seven VirB proteins of Agrobucterium tumefuciens:implications for the formation of a T-DNA transport structure. JBacteriol 17 5 5233-5241Thorstenson YR, Zambryski PC (1994) The essential virulenceprotein VirB8 localizes to the inner membrane of Agrobucteriumtumefaciens. J Bacteriol 176 1711-1717Tinland B, Hohn B, Puchta H (1994) Agrobucterium tumefucienstransfers single-stranded transferred DNA (T-DNA) into theplant cell nucleus. Proc Natl Acad Sci USA 91: 8000-8004Tinland B, Koukolikova NZ, Hall MN, Hohn B (1992) The T-DNA-linked VirD2 protein contains two distinct functional nu-clear localization signals. Proc Natl Acad Sci USA 89: 7442-7446

Waters VL, Guiney DG (1993) Processes at the nick region linkconjugation, T-DNA transfer and rolling circle replication. Mo1Microbiol 9: 1123-1 130Weiss AA, Johnson FD, Burns DL (1993) Molecular characteriza-tion of an operon required for pertussis toxin secretion. ProcNatl Acad Sci USA 9 0 970-2974

West SC (1992) Enzymes and molecular mechanisms of homolo-gous recombination. Annu Rev Biochem 61: 603-640Whiteside ST, Goodbourn S (1993) Signal transduction and nu-clear targeting: regulation of transcription factor activity bysubcellular localisation. J Cell Sci 104 949-955Winans SC (1992) Two-way chemical signaling in Agrobucterium-plant interactions. Microbiol Rev 56: 12-31

Yusibov VM, Steck TR, Gupta V, Gelvin SB (1994) Association ofsingle-stranded transferred DNA from Agrobacterium tumefacienswith tobacco cells. Proc Natl Acad Sci USA 91: 2994-2998Zambryski PC (1992) Chronicles from the Agrobacterium-plant cellDNA transfer story. Annu Rev Plant Physiol Plant Mo1 Biol 43:465-490

267: 20471-20480

321-324

69: 605-613

2925-2929

Transfer of T-DNA From Agrobacterium to the Plant Cell

Documents

Transcript of Transfer of T-DNA From Agrobacterium to the Plant Cell