Agrobacterium Tumefaciens Ti Plasmid Research Articles

Tobacco plants carrying a tms locus of Ti-plasmid origin and the HI-1 allele are tumor prone

The autonomous growth of plant tumor cells is believed to result from their persistent loss of the requirement for growth hormones such as auxin and cytokinin. The partially dominant gene Habituated leaf-1 (HI-1) regulates the requirement of cultures tissues of Havana 425 tobacco ( Nicotiana tabacum L.) for cytokinins. The HI-1 allele can partially restore the tumor phenotype in tobacco cells transformed with a Agrobacterium tumefaciens Ti plasmid defective in the isopentenyl transferase locus, which encodes a key enzyme in cytokinin biosynthesis and is required for neoplastic growth. To investigate the oncogenic function of HI-1, we transformed wild-type ( hl-1/hl-1) and Hl-1/Hl-1 tobacco plants with the tms locus derived from the limited-host-range Ti plasmid pTiAg162. This locus encodes enzymes for biosynthesis of the auxin indole-3-acetic acid. Grafting tests and measurements of the hormone requirement of cultured explants show that wound-induced overgrowths arising in tms transformed Hl-1 plants are tumorous. While some wound-induced overgrowths also formed in hl-1/hl-1 transformants, these showed slight hormone-autotrophic growth and weak tumorigenicity in grafting tests. In addition, Hl-1/Hl-1 tms/tms plants, but not hl-1/hl-1 tms/tms plants, spontaneously developed rooty teratomatous overgrowths, showed flowering abnormalities, and formed calli at the base of the stem in young seedlings. Thus, Hl-1 tms plants exhibit a tumor-prone phenotype, and in this regard closely resemble tumor-prone hybrids that arise in certain interspecific crosses of Nicotiana species. Our results show that the interaction of just two genetic elements – the mutant Hl-1 allele of the tobacco host with tms genes of Ti plasmid origin – are sufficient for a tumor-prone phenotype.

Detection and Enumeration of a Tagged Pseudomonas fluorescens Strain by Using Soil with Markers Associated with an Engineered Catabolic Pathway

Previously we described a novel gene tagging method, using the moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955, to identify microorganisms destined for release into the environment. Here, we used the engineered strain Pseudomonas fluorescens PF5MT12 carrying the moc region integrated into the bacterial chromosome to demonstrate the usefulness of the markers for detection and direct selection of marked organisms present in soil samples. Using this system, we routinely detected population levels as low as 10(sup2) CFU per g of soil sampled. In addition to direct selection, we developed an immunologically based assay using MOP cyclase, a unique enzyme associated with moc, as the epitope for detecting the tagged organism. The colony immunoblot assay proved to be highly specific and without any false-positive signals when used to identify organisms cultured from soil on nonselective medium. The numbers of colonies that were immunoreactive with the anti-MOP cyclase antibody were essentially equal to those that grew out on selection plates. This indicates that MOP cyclase can be used as a marker and that we can use nonselective medium to retrieve the marked genetically engineered microorganisms and then identify them by using colony immunoblot assays. These direct selection and colony immunoblot methods provide a sensitive and accurate strategy for identifying and enumerating marked organisms recovered from soil samples. We also developed a rapid assay for MOP cyclase that does not require cell permeabilization with toluene. This assay can be used to verify tagged organisms isolated by other methods or to screen large numbers of colonies for the tag following nonselective isolation.

The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F

The Ti plasmids of Agrobacterium tumefaciens encode two transfer systems. One mediates the translocation of the T-DNA from the bacterium to a plant cell, while the other is responsible for the conjugal transfer of the entire Ti plasmid from one bacterium to another. The determinants responsible for conjugal transfer map to two regions, tra and trb, of the nopaline-type Ti plasmid pTiC58. By using transposon mutagenesis with Tn3HoHo1, we localized the tra determinants to an 8.5-kb region that also contains the oriT region. Fusions to lacZ formed by transposon insertions indicated that this region is expressed as two divergently transcribed units. We determined the complete nucleotide sequence of an 8,755-bp region of the Ti plasmid encompassing the transposon insertions defining tra. The region contains six identifiable genes organized as two units divergently transcribable from a 258-bp inter-genic region that contains the oriT site. One unit encodes traA, traF, and traB, while the second encodes traC, traD, and traG. Reporter insertions located downstream of both sets of genes did not affect conjugation but were expressed, suggesting that the two units encode additional genes that are not involved in transfer under the conditions tested. Proteins of the predicted sizes were expressible from traA, traC, traD, and traG. The products of several Ti plasmid tra genes are related to those of other conjugation systems. The 127-kDa protein expressed from traA contains domains related to MobA of RSF1O1O and to the helicase domain of TraI of plasmid F. The translation product of traF is related to TraF of RP4, and that of traG is related to TraG of RP4 and to VirD4 of the Ti plasmid T-DNA transfer system. Genetic analysis indicated that at least traG and traF are essential for conjugal transfer, while sequence analysis predicts that traA also encodes an essential function. traB, while not essential, is required for maximum frequency of transfer. Patterns of sequence relatedness indicate that the oriT and the predicted cognate site-specific endonuclease encoded by traA share lineage with those of the transfer systems of RSF1010 and plasmid F, while genes of the Ti plasmid encoding other essential tra functions share common ancestry with genes of the RP4 conjugation system.

Purification and characterization of catabolic mannopine cyclase encoded by the Agrobacterium tumefaciens Ti plasmid pTi15955

Catabolic mannopine (MOP) cyclase encoded by certain Agrobacterium Ti and Ri plasmids lactonizes MOP to agropine (AGR). The enzyme, purified to homogeneity from a recombinant clone, has a molecular mass of 45 kDa as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size exclusion chromatography. The enzyme catalyzed the lactonization of MOP to AGR without the need for any cofactors. The enzyme also converted AGR to MOP with the lactonizing activity being predominant over the reverse reaction. MOP cyclase is specific for imine conjugates of D-hexose and L-glutamine and was not inhibited by sugars or amino acids. The enzyme lactonized deoxyfructosyl glutamine, a natural intermediate of MOP synthesis and catabolism, to a product indistinguishable from chrysopine, a newly discovered crown gall opine. The enzyme also lactonized N-l-(1,2-dideoxy-D-mannityl)-L-glutamine, indicating that a hydroxyl group at carbon atom 2 of the sugar moiety is not required for the enzymatic reaction.

Conserved cis-acting promoter elements are required for density-dependent transcription of Agrobacterium tumefaciens conjugal transfer genes.

Ti plasmids of Agrobacterium tumefaciens, in addition to transferring oncogenic DNA to the nuclei of infected plant cells, can conjugally transfer between agrobacteria. Conjugation of wide-host-range octopine-type Ti plasmids requires a tumor-released arginine derivative called octopine. Octopine stimulates expression of the traR gene, whose product directly activates other tra genes in the presence of an acylated homoserine lactone called Agrobacterium autoinducer (AAI). We have localized the transcription starts of three tra promoters and find conserved elements (tra boxes) at virtually identical positions upstream of each promoter. Disruption of these tra boxes abolished induction of each promoter. Deletion analysis of the traI promoter indicates that tra boxes are the only upstream elements required for transcriptional activation. Since Ti plasmid donor cells both produce and respond to AAI, we tested whether expression of tra promoters was enhanced by high concentrations of bacteria. Both tra gene expression and conjugation itself were strongly stimulated either by high donor densities or by exogenous AAI.

Transfer of non-T-DNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA.

We introduced a plant selection marker, nptII, to the left of border A in the Agrobacterium Ti plasmid pTiA6. Infection of tobacco leaf discs with the modified Agrobacterium strain gave rise to kanamycin-resistant calli which grew in a hormone-dependent manner. Southern hybridization analysis of DNA isolated from four transformants indicated initiation of DNA transfer at or near border A and absence of T-DNA sequences. These results demonstrate that DNA transfer events starting at a left border on a native Ti plasmid and moving away from the T-DNA region occur and that they can be detected by designing a suitable selection strategy.

An Agrobacterium virulence factor encoded by a Ti plasmid gene or a chromosomal gene is required for T-DNA transfer into plants.

Mutagenesis of the vir region on the Ti plasmid of Agrobacterium tumefaciens revealed a new locus, virJ, that is induced by the plant-wound signal molecule, acetosyringone (AS). virJ lies between virA and virB, and is transcribed in the same direction. The amino acid sequence of virJ is similar to a region of a previously characterized chromosomal gene, acvB, required for virulence. virJ can complement the avirulent phenotype of an acvB mutant, indicating that virJ and acvB encode the same factor required for tumorigenesis. Southern analysis revealed that virJ is present on the Ti plasmid of an octopine but not a nopaline strain whereas acvB is present on the chromosomes of both octopine and nopaline strains. While virJ is regulated by AS under the control of the virA/virG two-component regulatory system, acvB is not induced by AS. VirJ possesses a putative signal peptide and was found predominantly in the periplasmic fraction. The strain lacking both acvB and virJ had an impaired ability to transfer T-DNA into plant cells, suggesting that the factor encoded by virJ or acvB is required for T-DNA transfer from A. tumefaciens to plant cells. acvB is the first chromosomal gene implicated in T-DNA transfer, but whether it functions specifically for this process is not clear. We hypothesize that virJ evolved from acvB, presumably for a more specialized role in tumorigenesis.

Divergent Transcription and a Remote Operator Play a Role in Control of Expression of a Nopaline Catabolism Promoter in Agrobacterium tumefaciens

The nocP-nocR divergent gene arrangement of the nopaline catabolism (noc) operon of the Agrobacterium tumefaciens Ti plasmid pTiT37 was examined with respect to the expression of the nocP promoter. Under repressive conditions, i.e. in the absence of nopaline, four distinct levels of PnocP expression were observed. The lowest level of expression, i.e. full repression, was detected in the presence of the NocR repressor, together with the remote noc operator and productive transcription from the divergent nocR promoter. The next level was observed in the absence of either the NocR protein or of the operator or of both. The third level was detected when abortive transcription from the nocR promoter occurred, irrespective of the presence or absence of the NocR protein. The highest level of PnocP expression was observed in the absence of both productive transcription from PnocR and the operator sequence, whether or not the NocR protein was present. Under inductive conditions, i.e. in the presence of nopaline, expression of PnocP was activated if both the NocR protein and the operator were present. Absence of either NocR or the operator resulted in lack of inducibility of the nocP promoter. Transcription from the divergent nocR promoter had no influence on the activation of PnocP. It was also found that the absence of the operator affected plasmid supercoiling in vivo. The results suggest that DNA topology has a role in the regulation of the nocP promoter.

Common ancestry between IncN conjugal transfer genes and macromolecular export systems of plant and animal pathogens.

The DNA sequence of a cluster of pKM101 conjugal transfer genes was determined and aligned with the genetic map of the plasmid. Eighteen genes were identified, at least eight and probably 11 of which are required for efficient conjugation. These tra genes are homologous to and colinear with genes found in the virB operon of Agrobacterium tumefaciens Ti plasmids. Seven pKM101 tra genes are also homologous to ptl genes of Bordetella pertussis, which direct the export of pertussis toxin. We used TnphoA to construct translational fusions between pKM101 genes and the Escherichia coli phoA gene, which encodes alkaline phosphatase, and provide evidence that at least 11 of the 18 genes are either fully or partially exported from the cytoplasm.

Octopine and nopaline oxidases from Ti plasmids of Agrobacterium tumefaciens: molecular analysis, relationship, and functional characterization

The occ and noc regions of pTiAch5 (octopine) and pTiC58 (nopaline) Ti plasmids are responsible for the catabolic utilization of octopine and nopaline in Agrobacterium spp. The first enzymatic step is the oxidative cleavage into L-arginine and pyruvate or 2-ketoglutarate, respectively, by membrane-bound opine oxidases requiring two polypeptides (subunits B and A) for function. The DNA sequences showed that the subunits of pTiAch5 and pTiC58 are related, but none of the proteins revealed significant similarities to the biosynthetic enzymes expressed in transformed plant cells. The four proteins had no extensive overall similarity to other proteins, but the 35 N-terminal amino acids contained motifs found in many enzymes utilizing flavin adenine dinucleotide, flavin mononucleotide, or NAD(P)+ as cofactors. However, the activities were completely independent of added cofactors, and the nature of the electron acceptor remained unclear. Membrane solubilization led to complete loss of enzyme activity. The nopaline oxidase accepted nopaline and octopine (Vmax ratio, 5:1) with similar Km values (1.1 mM). The octopine oxidase had high activity with octopine (Km = 1 mM) and barely detectable activity with nopaline. The subunits from the occ and the noc regions were exchangeable. The combinations ooxB-noxA and noxB-ooxA both produced active enzymes which oxidized octopine and nopaline at similar rates, suggesting that both subunits contributed to the substrate specificity. These experiments also showed that the formation of functional enzyme required close proximity of the subunit genes on the same plasmid and that even a reversal of the gene order (A-B instead of B-A) led to reduced activity.

IPTASE SYNTHESIS AND LIGHT MEDIATION IN TRANSGENIC TOBACCO

Similarities exist between the effects of phytochrome and cytokinins on plant growth and development (e.g., chloroplast development, amaranthin synthesis. seed germination, photomorphogenesis). It is unclear, however, if and how these two systems interact. As a beginning step to determine cytokinin-phytochrome interactions, we developed a strategy utilizing ipt -transgenic tobacco in phytochrome/light treatment investigations. The sour-cc of the ipt gene was Agrobacterium tumefaciens Ti plasmid 15955. This gene encodes for isopentenyl transferase which is an enzyme active in cytokinin biosynthesis. Ipt -transgenic tobacco cultures (grown on MS medium supplemented with kanamycin but no plant growth regulators) were treated with end-of-day red or far-red light for 15 minutes. After 30 days of treatment, the plant tissue was harvested and either homogenized for SDS-PAGE or fixed for transmission electron microscopic analysis. Results from immuno-gold labelling using polyclonal antibodies specific to iptase will he used to Indicate the influence of phytochrome on cytokinin activity. Also, structural changes at the ultra-cellular level will be determined.

Association of single-stranded transferred DNA from Agrobacterium tumefaciens with tobacco cells.

During the inception of crown gall tumorigenesis, the transferred DNA (T-DNA) is processed from the Ti (tumor inducing) plasmid of Agrobacterium tumefaciens and is transferred to plant cells. T-DNA processing and transfer require the induction of vir (virulence) genes by phenolic compounds secreted by wounded plant cells. After vir gene induction, both single-stranded (T-strands) and double-stranded forms of processed T-DNA accumulate in the bacteria. Although current models favor the transfer of T-strands to plants, there has yet been no experimental evidence to show this. In this paper, we show that T-strands disappear from acetosyringone-induced A. tumefaciens within 30 min of bacterial cocultivation with tobacco protoplasts. PCR analysis of T-DNA associated with protoplasts indicates that single-stranded, but not double-stranded, T-DNA can be detected in the plant cells within 30 min of bacterial cocultivation. Control experiments show that this T-DNA does not originate from lysed contaminating bacterial cells. T-DNA transfer depends on a functional bacterial virB operon. Protoplast infections using an A. tumefaciens virE mutant result in a low level of accumulation of T-strands in the plant cells.

A novel gene tag for identifying microorganisms released into the environment

A novel method using a moc (mannityl opine catabolism) region from the Agrobacterium tumefaciens Ti plasmid pTi15955 was developed as a tag to identify genetically modified microorganisms released into the environment. Pseudomonas fluorescens 1855.344, a plant-growth-promoting rhizosphere bacterium, was chosen as the organism in which to develop and test the system. moc genes carried by pYDH208, a cosmid clone containing a 20-kb segment of the octopine-mannityl opine-type Ti plasmid, conferred on P. fluorescens strains the capacity to utilize mannopine and agropine (AGR) as a sole source of carbon and energy. Modified P. fluorescens strains containing moc or moc::nptII inserted into a chromosomal site were constructed by marker exchange. One such modified strain, PF5MT12, utilized AGR as a sole carbon source and contained detectable levels of mannopine cyclase, an easily assayable enzyme encoded by the moc region. Catabolism of AGR could be used to recover selectively the marked strain from mixed populations containing a large excess of closely related bacteria. Nucleic acid-based detection strategies were developed on the basis of the unique fusion region between Agrobacterium DNA and Pseudomonas DNA in strain PF5MT12. The specificity and sensitivity of detection of PF5MT12 were enhanced by amplifying the fused DNA region by using PCR. The target fragment could be detected at levels of sensitivity comparable to those of other described PCR-based gene tags, even in the presence of high levels of Agrobacterium, Pseudomonas, or Escherichia coli DNA. This gene tag strategy gives a method for direct selection and enumeration of the marked strain from mixtures containing a large excess of closely related bacteria and a sensitive and highly specific system for detection by PCR amplification of the target fragment even in the presence of large amounts of DNA from related or unrelated organisms.

α-Domain of human metallothionein IA can bind to metals in transgenic tobacco plants

With a view to exploring its use as a metal-binding factor in transgenic plants we prepared the alpha-domain of metallothionein by reconstitution of rabbit apometallothionein and proteolysis of MT-1 and MT-2 with subtilisin. The isolated alpha-domains were characterised by UV and CD spectroscopy Double-Stranded. DNA encoding the alpha-domain (106 bp) of the human MT-IA was constructed from chemically synthesized oligomers by repair synthesis and enzymatic ligation, cloned into pUC19 and sequenced. A expression construct containing the cloned alpha-domain was introduced into tobacco cells on a disarmed Agrobacterium tumefaciens Ti-plasmid. Transformed tobacco cells were selected and regenerated on medium containing cadmium and kanamycin. The growth of roots and shoots of transformants was unaffected by up to 100 microM cadmium, whereas control plants showed severe inhibition of root and shoot growth, and chlorosis of leaves on medium containing only 10 microM cadmium. Southern hybridization confirmed the presence of the transgene in the transformed plant tissues. The concentration of human alpha-domain peptides in transgenic tobacco leaves was determined by the Cd/hemoglobin saturation assay and polarography using the rabbit alpha-domain as standard. The results indicate that the alpha-domain, one of two domains in MT molecules, is not only stable in vitro, but is also expressed efficiently and functions independently in transgenic plant cells.

Genetic Organization of the Conjugal DNA Processing Region of the IncW Plasmid R388

The region of the IncW plasmid R388 involved in conjugal DNA metabolism and mobilization (MOB w) has been analyzed by Tn5tacl insertion mutagenesis, genetic complementation and DNA sequencing. Three genes ( trwA, trwB and trwC) were mapped within MOB w. They are transcribed from the same strand and away from oriT. The predicted products of trwA, trwB and trwC are proteins of 121, 507 and 966 amino acids, respectively. The three proteins were visualized in a minicell expression system, showing apparent molecular masses of 13·5, 55 and 105 kDa, respectively. The deduced amino acid sequence of TrwA shows significant similarity to TraJ of the IncP plasmids RP4 and R751, to NikA of the IncI plasmid R64 and to MobB of plasmid pTF-FC2. The amino acid sequence of TrwB predicts an integral membrane protein which contains an NTP-binding motif. It shows 28% to 29% identity with TraD of plasmids F and R100, 23% identity with TraG of plasmids RP4 and R751 and 20% identity with VirD4 of the Ti plasmids of Agrobacterium tumefaciens. The amino acid sequence of TrwC shows the characteristic motifs of the Rep family of DNA helicases, It shows 33% identity with the sequence of helicase I (TraI) of plasmid F. The similarity is highest in the N-terminal segments of the proteins, which show conservation of characteristic amino acid motifs of a family of DNA-relaxases, including VirD2 of the Ti plasmid. The conserved features of these three proteins among the different transfer systems suggest that a very widespread conjugal DNA mobilization mechanism is shared by the transfer apparatuses of IncF, IncI, IncP, IncW and Ti plasmids.

Fruit‐specific expression of the A. tumefaciens isopentenyl transferase gene in tomato: effects on fruit ripening and defense‐related gene expression in leaves

SummaryThis paper describes the analysis of tomato plants transformed with a chimeric gene consisting of the promoter region of a fruit specifically expressed tomato gene linked to the ipt gene coding sequences from the Ti plasmid of Agrobacterium tumefaciens. The pattern of expression of this chimeric gene was found to be consistent with the expression of the endogenous fruit‐specific gene and consequently, plants expressing the chimeric gene were phenotypically normal until fruit maturation and ripening. A dramatically altered fruit phenotype, islands of green pericarp tissue remaining on otherwise deep red ripe fruit, was then evident in many of the transformed plants. Cytokinin levels in transformed plant fruit tissues were 10 to 100‐fold higher than in control fruit. In the leaves of a fruit‐bearing transformant, despite a lack of detectable ipt mRNA accumulation, approximately fourfold higher than control leaf levels of cytokinin were detected. It is suggested that cytokinin produced in fruit is being transported to the leaves since accumulation in leaves of PR‐1 and chitinase mRNAs, which encode defense‐related proteins known to be induced by cytokinin, occurred only when the transformant was reproductively active. Effects of elevated cytokinin levels on tomato fruit gene expression and cellular differentiation processes are also described.

Site-specific cleavage and joining of single-stranded DNA by VirD2 protein of Agrobacterium tumefaciens Ti plasmids: analogy to bacterial conjugation.

As an early stage of plant transformation by Agrobacterium tumefaciens, the Ti plasmid is nicked at the border sequences that delimit the T-DNA. Cleavage results in covalent attachment of VirD2 to the 5' terminal of the nicked strand by a process resembling initiation of DNA transfer that occurs in the donor cell during bacterial conjugation. We demonstrate that this cleavage can be reproduced in vitro: VirD2 protein, the border-cleaving enzyme, was overproduced and purified. Cleavage assays were performed with single-stranded oligodeoxyribonucleotides encompassing the Ti plasmid border region or the transfer origin's nick region of the conjugative plasmid RP4. VirD2 of pTiC58 cleaves both border- and nick region-containing oligonucleotides. However, the relaxase TraI of RP4 can cut only the cognate nick regions. The respective proteins remain covalently bound to the 5' end of the cleavage sites, leaving the 3' termini unmodified. VirD2-mediated oligonucleotide cleavage was demonstrated to be an equilibrium reaction that allows specific joining of cleavage products restoring border and nick regions, respectively. The possible role of VirD2 in T-DNA integration into the plant cell's genome is discussed in terms of less stringent target-sequence requirements.

Nopaline causes a conformational change in the NocR regulatory protein-nocR promoter complex of Agrobacterium tumefaciens Ti plasmid pTiT37

The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein. The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins. Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline. The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex. Sequence analysis of the NocR binding site indicated the presence immediately downstream of the -10 sequence of the nocR promoter of a 12 bp putative operator overlapping a consensus gyrase recognition sequence and an 18 bp long alternating purine-pyrimidine sequence. These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.

Replication of tomato yellow leaf curl virus (TYLCV) DNA in agroinoculated leaf discs from selected tomato genotypes.

The leaf disc agroinoculation system was applied to study tomato yellow leaf curl virus (TYLCV) replication in explants from susceptible and resistant tomato genotypes. This system was also evaluated as a potential selection tool in breeding programmes for TYLCV resistance. Leaf discs were incubated with a head-to-tail dimer of the TYLCV genome cloned into the Ti plasmid of Agrobacterium tumefaciens. In leaf discs from susceptible cultivars (Lycopersicon esculentum) TYLCV single-stranded genomic DNA and its double-stranded DNA forms appeared within 2-5 days after inoculation. Whiteflies (Bemisia tabaci) efficiently transmitted the TYLCV disease to tomato test plants following acquisition feeding on agroinoculated tomato leaf discs. This indicates that infective viral particles have been produced and have reached the phloem cells of the explant where they can be acquired by the insects. Plants regenerated from agroinfected leaf discs of sensitive tomato cultivars exhibited disease symptoms and contained TYLCV DNA concentrations similar to those present in field-infected tomato plants, indicating that TYLCV can move out from the leaf disc into the regenerating plant. Leaf discs from accessions of the wild tomato species immune to whitefly-mediated inoculation, L. chilense LA1969 and L. hirsutum LA1777, did not support TYLCV DNA replication. Leaf discs from plants tolerant to TYLCV issued from breeding programmes behaved like leaf discs from susceptible cultivars.

Genetic analysis of the agrocinopine catabolic region of Agrobacterium tumefaciens Ti plasmid pTiC58, which encodes genes required for opine and agrocin 84 transport.

The acc region, subcloned from pTiC58 of classical nopaline and agrocinopine A and B Agrobacterium tumefaciens C58, allowed agrobacteria to grow using agrocinopine B as the sole source of carbon and energy. acc is approximately 6 kb in size. It consists of at least five genes, accA through accE, as defined by complementation analysis using subcloned fragments and transposon insertion mutations of acc carried on different plasmids within the same cell. All five regions are required for agrocin 84 sensitivity, and at least four are required for agrocinopine and agrocin 84 uptake. The complementation results are consistent with the hypothesis that each of the five regions is separately transcribed. Maxicell experiments showed that the first of these genes, accA, encodes a 60-kDa protein. Analysis of osmotic shock fractions showed this protein to be located in the periplasm. The DNA sequence of the accA region revealed an open reading frame encoding a predicted polypeptide of 59,147 Da. The amino acid sequence encoded by this open reading frame is similar to the periplasmic binding proteins OppA and DppA of Escherichia coli and Salmonella typhimurium and OppA of Bacillus subtilis.