Virulence of Agrobacterium tumefaciens Strain A281 on Legumes

This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an l,l-succinamopine strain. We tested virulence of T-DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid regions from heterologous nopaline and octopine strains.     

Detection of T-DNA transfer to plant cells by A. tumefaciens virulence mutants using agroinfection

Summary To test whether virulence mutants of Agrobacterium tumefaciens are capable of promoting T-DNA transfer into plant cells, a tandem array of Cauliflower Mosaic Virus (CaMV) DNA was cloned between T-region border sequences on a wide host range plasmid and introduced into various virulence mutants. The resulting strains were used to infect Brassica rapa cv. Just Right. This assay, recently referred to as agroinfection, is based on the appearance of viral symptoms following transfer of T-DNA to plant cells, and is shown to be at least 100 times more sensitive in detecting T-DNA transfer than tumour formation. Mutants in the loci vir A, B and G, which were avirulent on turnip, failed to induce virus symptoms. Of the two vir D mutants tested, neither induced tumours, but one was capable of inducing virus symptoms. Mutants in vir E, C and F, which induced respectively no, small and normal tumours on turnip, all induced virus symptoms.     

Nucleotide sequence, evolutionary origin and biological role of a rearranged cytokinin gene isolated from a wide host range biotype III Agrobacterium strain

Summary A DNA fragment with homology to the cytokinin (ipt) gene from biotype I Agrobacterium tumefaciens strain Ach5 was cloned from the Ti plasmid of the wide host range biotype III Agrobacterium strain Tm-4 and sequenced. The fragment contains an intact ipt coding sequence. However, the 3 non-coding region of this ipt gene is rearranged due to a 0.9 kb deletion fusing it to the 3 coding region of the neighbouring gene 6a, most of which was found to be deleted. The Tm-4 ipt gene is strongly related to the partially deleted ipt gene of the limited host range biotype III strain Ag162. To test its biological activity, the Tm-4 ipt gene was inserted into a specially constructed, disarmed Ti vector lacking tzs and tested on tobacco, where the rearranged ipt gene induced shoot formation. The cloned Tm-4 ipt gene was mutated with Tn5 and the intact gene on the wild-type Tm-4 Ti plasmid was replaced by the mutated gene. The resulting strain was avirulent on tobacco but normally virulent on the natural host of the wild-type strain Tm-4, grapevine. As the biotype 1 6b gene diminishes the effect of a corresponding ipt gene, a larger Tm-4 fragment carrying both the ipt gene and an adjacent 6b-like gene was also tested on tobacco and compared with the Tm-4 ipt fragment alone and with an ipt and 6b/ipt fragment derived from Ach5. The Tm-4 6b gene diminishes the effect of the Tm-4 ipt gene, showing the Tm-4 6b gene to be active as well. The Tm-4 6b/ipt combination is less effective than the Ach5 combination. These results provide further insight into the molecular basis of the host range differences between limited host range and wide host range biotype III Agrobacterium strains and show that the WHR cytokinin gene, although active, does not significantly contribute to tumour formation on the natural host of the WHR biotype III strains, grapevine.Abbreviations LHR limited host range - WHR wide host range - onc oncogenicity genes - iaaH indoleacetamide hydrolase gene - iaaM tryptophan monooxygenase gene - ipt isopentenyl transferase gene - tzs transzeatin secretion gene - NAA -naphthalene acetic acid - BAP 6-benzylaminopurine - Km kanamycin - Neo neomycin - Cm chloramphenicol     

Analysis of conditions forAgrobacterium-mediated transformation of tobacco cells in suspension

We have developed anAgrobacterium-mediated transformation system, using tobacco cell suspensions, that permits evaluation of factors affecting transformation within seven days of co-cultivation. Tobacco cell transformation was determined by monitoring -glucuronidase (GUS) activity detected in plant cell extracts. The use of a chimeric gene construct, 35S-GUS/INT, containing a portable intron in theuidA reading frame, assured only plant-specific GUS expression. During the co-cultivation period, induction of the bacterialvir-region was monitored using a heterologous gene construct composed of avirB promoter fragment from pTiC58 fused to the chloramphenicol acetyltranferase (CAT) gene ofTn9. Tobacco cell transformants were confirmed by antibiotic selection of transformed plant cells and by X-Gluc staining. Maximum transformation was obtained when plant suspension cultures were growing rapidly which also was coincidental with elevated levels of bacterialvir-region expression. One week after co-cultivation, the transformed cultures exhibited a stable pattern of GUS activity which remained constant without antibiotic selection. The system was used to compare the virulence of a number ofAgrobacterium strains. GUS activity of plant cells co-cultivated with a strain containing a cointegrate plasmid was 3-fold higher than that of one with a binary configuration of the T-DNA. When the co-cultivatingAgrobacterium strain also carried the plasmid used to monitorvir induction, the frequency of transformation was reduced by as much, as 97%.     

A partially disarmed<Emphasis Type="Italic"> vir</Emphasis> helper plasmid,pKYRT1, in conjunction with 2,4-dichlorophenoxyactic acid promotes emergence of regenerable transgenic somatic embryos from immature cotyledons of soybean

Agrobacterium tumefaciens strain KYRT1 harboring the virulence helper plasmid pKYRT1 induces transgenic somatic embryos (SEs) at high frequency from infected immature soybean cotyledons. KYRT1 is derived from the highly oncogenic strain Chry5. However, pKYRT1 is not completely disarmed and still contains an entire T-right (T_R) and a portion of T-left (T_L). In this report, binary strains, each carrying fully disarmed vir helper plasmids including pKPSF2, which is a fully disarmed version of pKYRT1, were compared to strain KYRT1 for their ability to induce transgenic SEs on immature cotyledons of soybean. Six weeks following cocultivation, histochemical GUS assays of cultured explants indicated that all fully disarmed vir helper plasmids transferred their binary T-DNA, containing a GUS-intron gene, into soybean tissues. However, none of these transformed tissues developed SEs on medium with or without 2,4-dichlorophenoxyactic acid (2,4-D). On the other hand, immature cotyledons cocultivated with strain KYRT1 exhibited high induction of transgenic SEs, but only on medium supplemented with 2,4-D. Derivatives of strain Chry5 harboring other vir helper plasmids did not induce transgenic SEs under any conditions tested, thus suggesting that the chromosomal background of KYRT1 alone was not sufficient to promote somatic embryogenesis. PCR analysis indicated that 55% of transgenic embryogenic cultures and 29% of transgenic T₀ soybean plants derived by transformation using strain KYRT1 contained T_R from pKYRT1 in addition to the uidA gene from the binary construct. None of the transgenic tissues or T₀ plants contained T_L DNA. These results suggest that some function coded for by T_R of pKYRT1 influences somatic embryogenesis in conjunction with exposure of the plant tissues to 2,4-D. Since the co-transformation frequency of the undesirable T-DNA sequences from the vir helper plasmid was relatively low, the partially disarmed strain KYRT1 will likely be very useful for the production of normal transgenic plants of diverse soybean cultivars.Abbreviations 2,4-D 2,4-Dichlorophenoxyactic acid - GUS -Glucuronidase - hpt Hygromycin phosphotransferase gene - SE Somatic embryo - uidA -Glucuronidase gene     

The promoter proximal region in the virD locus of Agrobacterium tumefaciens is necessary for the plant-inducible circularization of T-DNA

Summary The formation of crown gall tumours involves the transfer of the T-DNA region of the Ti plasmid from Agrobacterium to plant cells and its subsequent integration into plant chromosomes. When agrobacteria are incubated with plant protoplasts or exudates of plants, the T-DNA region is circularized by recombination or cleavage and rejoining between the 25 bp terminal repeats; the formation of circular T-DNAs is thought to be one step in T-DNA transfer (Koukolikova-Nicola et al. 1985; Machida et al. 1986). We previously showed that the virulence region of the Ti plasmid is required for T-DNA circularization. In the present paper, we examined the circularization event in agrobacteria harbouring octopine Ti plasmids with mutations in various loci of the virulence region. The results clearly demonstrate that the gene(s) encoded in the virD locus are necessary for T-DNA circularization. In particular, the gene(s) present in the region proximal to the virD promoter are essential. We propose that roduct(s) of this gene have recombinase or endonuclease activity which specifically recognizes the 25 bp terminal repeats of T-DNA.     

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants

Transposon-insertion mutants with vir^? Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir^? nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir^? octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir^? octopine Ti plasmid mutations and vir^? nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO^? mutants produced rooty tumors on Kalanchoë tubiflora.     

Limited-host-range plasmid of Agrobacterium tumefaciens: molecular and genetic analyses of transferred DNA.

A tumor-inducing (Ti) plasmid from a strain of Agrobacterium tumefaciens that induces tumors on only a limited range of plants was characterized and compared with the Ti plasmids from strains that induce tumors on a wide range of plants. Whereas all wide-host-range Ti plasmids characterized to date contain closely linked oncogenic loci within a single transferred DNA (T-DNA) region, homology to these loci is divided into two widely separated T-DNA regions on the limited-host-range plasmid. These two plasmid regions, TA-DNA and TB-DNA, are separated by approximately 25 kilobases of DNA which is not maintained in the tumor. The TA-DNA region resembles a deleted form of the wide-host-range TL-DNA and contains a region homologous to the cytokinin biosynthetic gene. However, a region homologous to the two auxin biosynthetic loci of the wide-host-range plasmid mapped within the TB-DNA region. These latter genes play an important role in tumor formation because mutations in these loci result in a loss of virulence on Nicotiana plants. Furthermore, the TB-DNA region alone conferred tumorigenicity onto strains with an intact set of vir genes. Our results suggest that factors within both the T-DNA and the vir regions contribute to the expression of host range in Agrobacterium species. There was a tremendous variation among plants in susceptibility to tumor formation by various A. tumefaciens strains. This variation occurred not only among different plant species, but also among different varieties of plants within the same genus.     

The limited host range of an Agrobacterium tumefaciens strain extended by a cytokinin gene from a wide host range T-region

The host range of Agrobacterium tumefaciens strain LBA649 (pTiAg57) is limited to grapevine and a few other plant species. Its host range was extended through the introduction of the T-region from the wide host range octopine plasmid pTiAch5. In contrast, R prime plasmids harboring the entire wide host range virulence region were unable to achieve this effect. Via site-directed mutagenesis a search was performed to identify the T-DNA genes which were responsible for the observed host range extension. Inactivation of one of the onc-genes (the cyt gene) was found to abolish the capacity of the T-region to extend the host range of LBA649. Therefore, we cloned the cyt gene into a disarmed T-region plant vector and used it in complementation studies with pTiAg57 via the binary vector strategy. We show that the mere presence of the cyt gene from a wide host range Ti plasmid is sufficient to extend the host range of LBA649 to certain plants. We conclude that the limited host range of LBA649 is not caused by a lack of recognition of plants but is mainly due to the absence or inactivity of a cyt gene in the T-region of pTiAg57.     

T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis

The soil bacterium Agrobacterium tumefaciens can transfer a part of its tumour-inducing (Ti) plasmid, the T-DNA, to plant cells. The virulence (vir) genes, also located on the Ti plasmid, encode proteins involved in the transport of T-DNA into the plant cell. Once in the plant nucleus, T-DNA is able to integrate into the plant genome by an illegitimate recombination mechanism. The host range of A. tumefaciens is not restricted to plant species. A. tumefaciens is also able to transfer T-DNA to the yeast Saccharomyces cerevisiae. In this paper we demonstrate transfer of T-DNA from A. tumefaciens to the yeast Kluyveromyces lactis. Furthermore, we found that T-DNA serves as an ideal substrate for gene targeting in K. lactis. We have studied the efficiency of gene targeting at the K. lactis TRP1 locus using either direct DNA transfer (electroporation) or T-DNA transfer from Agrobacterium. We found that gene targeting using T-DNA was at least ten times more efficient than using linear double-stranded DNA introduced by electroporation. Therefore, the outcome of gene targeting experiments in some organisms may depend strongly upon the DNA substrate used. Received: 11 May 1998 / Accepted: 16 October 1998     

Temperature-sensitive step in Ti plasmid vir-region induction and correlation with cytokinin secretion by Agrobacteria

Summary DNA transfer from Agrobacteria to plant cells requires activation of functions which are inactive under normal growth conditions. We studied two aspects with nopaline plasmid pGV3850: (1) conditions required for induction of a representative vir-region protein (virD2); for this we prepared antiserum against the protein and used the Western blot technique, and (2) correlation between vir-region induction and secretion of plant hormones. The results show that three factors are necessary and sufficient: the previously identified acetosyringone and acidic pH and, in addition, a carbon/energy source. Induction correlates with cytokinin secretion, suggesting that release of this hormone by the bacteria may play a role in tumor induction. No pronounced correlation is observed with release of indole-3-acetic acid. VirD2 induction and cytokinin secretion are temperature-dependent with similar optima. It is proposed that the thermosensitive step discovered decades ago with tumor induction in planta is in the activation of the vir functions.Abbreviations vir virulence gene - iP N⁶-(2-isopentenyl)adenine - iPA N⁶-(2-isopentenyl)adenosine - trans-Z trans-Zeatin - trans ZR, trans-Zeatinriboside - IAA indole-3-acetic acid - IPTG isopropyl--D-thiogalactoside     

Factors affecting the rate of T-DNA transfer from Agrobacterium tumefaciens to Nicotiana glauca plant cells

Different factors involved in the early steps of the T-DNA transfer process were studied by using a -glucuronidase gene (gusA) as a reporter in Nicotiana glauca leaf disc transformation experiments. The levels of transient expression of the gusA gene in leaf discs infected with several strains or vir mutants correlated well with their virulence phenotype, except for virC mutants. The rate of T-DNA transfer was shown to be stimulated in the case of non-oncogenic strains by the co-transfer of small amounts of oncogenic genes. It was found that the location of the T-DNA in the Agrobacterium genome affected the T-DNA transfer rate especially in virC mutants. The virC mutants transferred the gusA-containing T-DNA located on a binary vector more efficiently than the oncogenic T-DNA of the Ti plasmid. Although wild-type strains induced high levels of gusA expression early after infection, the gusA expression appeared to be lost late after infection in the infected leaf discs. In contrast, in leaf discs infected by virC mutants the level of gusA expression increased steadily in time. A model explaining these results is presented.     

A thin layer chromatographic technique for detecting inducers of Agrobacterium virulence genes in corn,wheat and rye

Summary A method is described for detecting plant metabolites capable of inducing the virulence genes of Agrobacterium tumefaciens. The method uses A. tumefaciens containing a plasmid with an inducible virulence gene fused to a galactosidase gene (virE::lacZ). Thin layer chromatography plates are overlayed with agar containing the indicator bacterium and a chromogenic galactoside (X-gal). Virulence gene inducing plant metabolites induce galactosidase which releases an aglycone readily oxidized by air to a blue pigmented zone at the Rf of the inducer. The method has been used to demonstrate the presence of virulence gene inducers in corn, wheat and rye. The uninduced background level of galactosidase also permits detection of bacterial growth inhibitors after a longer incubation period.Abbreviations X-gal 5-Bromo-4-chloro-3-indoxyl ß-D-galactopyranoside - TLC Thin Layer Chromatography - AS Acetosyringone - DIMBOA 2,4-Dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one - MBOA 6-Methoxy-2(3H)-benzoxazolone - T-DNA Transfer DNA - Ti Plasmid Tumor-inducing Plasmid     

Shorter T-DNA or additional virulence genes improve Agrobactrium-mediated transformation

The effect of additional virulence (vir) genes and size of plasmid T-DNA in Agrobacterium tumefa- ciens was investigated for their impact on transformation efficiency. Transformation efficiency in tobacco, cotton, and rice was increased when the T-DNA was 4.3 kb compared to 8.4 kb in size. However, when additional virG, virGN54D,virE, or virE/virG plasmids were included with the 8.4-kb T-DNA, transformation frequencies in all cases were increased over that of the shorter T-DNA without additional vir plasmids. The use of virE, virG or virGN54D copies enhanced transformation efficiency; however, the most significant increase of transformation efficiency in all three plant species was observed when the virE/virG plasmid was used for infection. The virE/virG plasmid dramatically enhanced the efficiency of Agrobacterium-mediated gene transfer; moreover, this plasmid appears to have broad efficiency since it was consistently effective on two different dicotyledon species as well as a monocotyledon species. Received: 8 February 2000 / Accepted: 21 March 2000     

Metabolic factors capable of inducing Agrobacterium vir gene expression are present in rice (Oryza sativa L.)

Summary The effects of exudates and extracts from suspension cultures or various parts of rice (Oryza sativa L.) plants on induction of vir (virulence) gene expression in Agrobacterium tumefaciens were examined. Only leaf extracts from panicle-differentiating plants to flowering plants were able to strongly induce activation and expression of vir genes. This induction was similar to that observed with 2 M acetosyringone (AS), yet there was no synergy between AS and rice extracts. Responses to vir-inducing metabolites and signal molecules were different among various vir loci. These results demonstrate that one or more inducing factors for vir gene expression are also present in rice, but only in specific parts and developmental stages.     

Genetic complementation of Agrobacterium tumefaciens Ti plasmid mutants in the virulence region

Summary Mutants with Tn5 insertions in the vir region of the Agrobacterium tumefaciens TiC58 plasmid are unable to form crown-gall tumors. Complementation tests of these vir region mutants were carried out by constructing merodiploids in a recombination-deficient strain. Each merodiploid possessed a mutant TiC58 plasmid and a recombinant plasmid containing either the homologous wild-type DNA region or the homologous region containing a second Tn5 insertion. The analysis identified six complementation groups. Mutations in one of these complementation groups were not complemented in trans and represent a cis-dominant locus. The mutation in one complementation group showed variation in host range.     

Absence in monocotyledonous plants of the diffusible plant factors inducing T-DNA circularization and vir gene expression in Agrobacterium

Summary T-DNA circularization is one of the molecular events specifically induced in agrobacterial cells upon their infection of dicotyledonous plant cells. We developed a seedling co-cultivation procedure to determine whether or not monocotyledonous plants have the ability to induce T-DNA circularization and vir gene expression. Co-cultivation of Agrobacterium tumefaciens with seedlings of dicotyledonous plants showed that the circularization event takes place efficiently. The exudates and extracts of the seedlings also effectively induced T-DNA circularization and vir gene expression, indicating that dicotyledonous seedlings contain diffusible factors capable of inducing these molecular events. In contrast, neither T-DNA circularization nor vir gene expression was detectable when Agrobacterium was incubated with seedlings of monocotyledonous plants. Supplementing with acetosyringone, a known inducer of vir gene expression and T-DNA circularization, resulted in the induction of circularization during co-cultivation with monocotyledonous seedlings. These results indicate that the seedlings of monocotyledonous plants have no detectable amounts of diffusible inducers, unlike dicotyledonous seedlings. Therefore, it is unlikely that the vir genes are expressed in Agrobacterium inoculated in monocotyledonous plants. This may be one of the blocks in tumorigenesis of monocotyledonous plants by Agrobacterium.     

Mechanisms of crown gall formation: T-DNA transfer fromAgrobacterium tumefaciens to plant cells

Agrobacterium tumefaciens harbouring the Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, T-DNA in the Ti plasmid is transferred by unknown mechanisms to plant cells to be integrated into nuclear DNA. WhenAgrobacterium is incubated with protoplasts or seedlings of dicotyledonous plants, circulation of T-DNA and expression ofvir (virulence) genes on the Ti plasmid are induced. The circularization event is efficiently induced by mesophyll protoplasts of tobacco which are highly competent for transformation by the T-DNA, and is also induced by diffusible phenolic compounds excreted from the protoplasts. The circularization and formation of crown gall both require the expression of thevirD locus, one of the induciblevir genes. These results suggest that the circularization of T-DNA reflects one of steps of the T-DNA transfer during formation of crown gall. In contrast to dicotyledonous plants, monocotyledonous plants are thought to be unresponsive to infection byAgrobacterium. We showed that monocotyledonous plants do not excrete diffusible inducers for the expression ofvir genes, while they contain a novel type of a signal substance(s). This inducer is not detected in the exudates of seedlings of monocotyledonous plants, but is found in the extracts from the seedlings, and also those from the seeds, bran and germ of wheat and oats. This finding suggests that T-DNA processing, and possibly its transfer, should take place whenAgrobacterium invades seedlings and seeds of monocotyledonous plants. Recipient of the Botanical Society Award for Young Scientists, 1987.     

Genetic analysis of the individual T-DNA genes of Agrobacterium tumefaciens; further evidence that two genes are involved in indole-3-acetic acid synthesis

Summary The T-DNA genes of Ti plasmids of Agrobacterium tumefaciens can induce tumorous growth on a wide range of dicotyledonous plants. We subcloned the individual onc genes of the pTiC58 T-DNA and reintroduced them in the T-region of the Ti plasmid gene vector pGV3850 (from which the onc genes had been removed (Zambryski et al. 1983)). These experiments were designed to analyze the contribution of each onc gene to the development of a tumor and have fulfilled two purposes. First, it was found that only the strains carrying gene 4 produced tumors without the aid of other T-DNA genes; in cell culture these tumors sprout shoots. Second, the shoot-forming phenotype of tumors induced by agrobacteria carrying Ti plasmids defective in either gene 1 or gene 2 can be restored to wildtype phenotype by simple coinfection with Agrobacterium strains whose Ti plasmids contain respectively only gene 2, or only gene 1 in their T-region. A parallel experiment demonstrated that the combined action of genes 1 and 2 is sufficient to induce tumor formation on tobacco plantlets.The external addition of -naphthalene acetic acid (NAA) restores to wild-type the phenotype of tumors induced by mutants in gene 1 or in gene 2. However, -naphthalene acetamide can only restore to wild-type the phenotype of mutants in gene 1. These data indicate that the product of the T-DNA gene 2 participates in the conversion of -naphthalene acetamide to a biologically active auxin, presumably NAA, and suggest that gene 1 codes for an enzyme involved in the synthesis of an indole-3-acetyl derivative.