Vol. 172, No. 4
JOURNAL OF BACTERIOLOGY, Apr. 1990, p. 1814-1822 0021-9193/90/041814-09$02.00/0 Copyright C) 1990, American Society for Microbiology
A Chromosomal Agrobacterium tumefaciens Gene Required for Effective Plant Signal Transduction MEEI-LI WU HUANG,lt GERARD A. CANGELOSI,2 WALTER HALPERIN,1 AND EUGENE W. NESTER2* Department of Botany1 and Department of Microbiology,2 University of Washington, Seattle, Washington 98195 Received 5 October 1989/Accepted 29 December 1989 The vir gene products of Agrobactenium tumefaciens carry out the transfer of T-DNA to the plant genome. Effective transcriptional induction of the vir genes by plant signal molecules is controlled by two vir gene products, VirA and VirG. In this study we have identified and cloned a chromosomal region which is also required for vir gene induction. Transposon insertions within this region reduce induction significantly and strongly attenuate virulence, resulting in a restricted host range for infection. The reduction in vir gene transcription can be partially overcome by high concentrations of the inducer molecule acetosyringone. Expression of virG at low pH and low phosphate concentrations, which is independent of plant signals, is not affected by these mutations. Sequence analysis of the region revealed two divergent open reading frames, which we have designated chvE and ORF1. Several transposon insertions mapped in chvE; this resulted in attenuated virulence. chvE codes for a putative protein which is homologous to two periplasmic receptor proteins involved in chemotaxis and uptake of sugars. Whether ORF1 is required for virulence is uncertain. One transposon insertion resulting in avirulence maps in or near the 5' end of ORF1, and several which do not affect virulence map in its 3' end. ORF1 codes for a putative protein which is homologous to a family of transcriptional activator proteins.
The host range for Agrobacterium infection is determined largely by the T-DNA and vir regions of the Ti plasmid (28, 29, 35, 50). Increasing the expression of certain vir genes increases the virulence and host range of the bacteria (25). However, several observations have indicated that chromosomal genes are also involved in determining host range (17, 20). In this study, we found that chromosomal host range mutants originally isolated by Garfinkel and Nester (17) are defective in vir gene induction.
Strains of Agrobacterium tumefaciens containing a Ti plasmid can incite crown gall tumors on most dicotyledonous and some monocotyledonous plants. Two regions of the Ti plasmid, the transferred DNA (T-DNA) and virulence (vir) regions, are necessary for tumor formation. During infection of wounded plant tissue, the T-DNA is transferred and integrated into the plant nuclear DNA (6, 49), where its expression results in crown gall tumor formation (12, 18, 53). In addition to the Ti plasmid virulence genes, chromosomal genes chvA, chvB, and exoC are required for complete virulence (3, 11). The functioning of these genes is required for attachment of the bacteria to plant cells. Transcription of the vir genes is induced by phenolic compounds, such as acetosyringone, synthesized by wounded plant cells (43, 44). The induction system is controlled by two of the vir genes, virA and virG (45, 54). virA codes for an inner membrane protein believed to detect the signal molecules, and virG codes for a cytoplasmic protein believed to be a transcriptional activator. Mutations in either virA or virG abolish both vir gene induction and virulence. Two chromosomal genes, ros and chvD, are also somehow involved in regulating vir gene expression. In ros mutants, expression of virC and virD genes is elevated even in the absence of inducers and polysaccharide synthesis is strongly repressed. However, the induction of all other vir genes is normal, and virulence is unaffected (7). In the absence of plant inducers, the expression of virG in wild-type strains is elevated by growth under acidic conditions and by phosphate starvation. This induction is influenced by chvD (55). Mutations in chvD attenuate both vir gene induction and virulence. How these two chromosomal loci influence vir gene expression is not clear. *
MATERIALS AND METHODS
Bacterial strains, plasmids, and media. Escherichia coli DH5(x, HB101 (1), and SF800 (22) were grown in LB liquid medium and on L agar (33), supplemented when appropriate with 100 ,ug of kanamycin per ml, 100 ,ug of carbenicillin per ml, 20 ,ug of tetracycline per ml, 50 ,ug of nalidixic acid per ml, or 100 ,ug of chloramphenicol per ml. A. tumefaciens strains (Table 1) were maintained on AB minimal medium and MG/L medium (5) supplemented when appropriate with 100 p,g of kanamycin per ml, 100 ,ug of carbenicillin per ml, or S pug of tetracycline per ml. vir gene induction assays. Plasmids carrying vir::lacZ fusions (Table 1) were introduced into the appropriate A. tumefaciens strains by triparental mating (9). Cells were cultured in either MS medium (34) or induction medium (55). When appropriate, acetosyringone (Aldrich Chemical Co., Inc.) was added to the concentrations indicated in the Results. At the times indicated, 1-ml samples were removed and frozen at -70°C. After the last samples were collected, all samples were thawed and 3-galactosidase activity was assayed as described by Stachel et al. (42). Molecular cloning. Total DNA from A1068 (Table 1) was partially digested with EcoRI and electrophoresed in an agarose gel. DNA of approximately 20 kilobases (kb) was electroeluted from the gel and ligated into the EcoRI site of
Corresponding author.
t Present address: Department of Genetics, University of Washington, Seattle, WA 98195. 1814
VOL. 172, 1990
AGROBACTERIUM CHROMOSOMAL VIRULENCE GENE
TABLE 1. Agrobacterium strains and plasmids used in this study Strain or plasmid
Strains A136 A348 A722
A723
A1038 A1008 A1059 A1068 A348MX1 A723MX1 MX557
Plasmids pSM243cd
pSW161 pSW162 pSM358cd
pSW174 pMWH102
pMWH112 pMWH146
Relevant genotype or phenotype'
C58 chromosome (Rif' Nalr), no Ti plasmid A136 with pTiA6NC A114 (C58 chromosome) with pTiA6NC A114 (C58 chromosome) with pTiB6806 A723, chvB::Tn5 (Kmr) A722, chvE::TnS (Kmr) A723, chvE::Tn5 (Kmr) A723, chvE::Tn5 (Kmr) A348, chvE::Tn5 (Kmr) A723, chvE::TnS (Kmr) A348, chvE::Tn3-HoHol (CbY) or ORF1::Tn3-HoHol (Cb')
virB::Tn3-HoHol (vir::lacZ fusion, Cbr) virC::Tn3-HoHol (vir::IacZ fusion, Cb`) virD::Tn3-HoHol (vir::IacZ fusion, Cb') virE::Tn3-HoHol (vir::lacZ fusion, Cbr) virG::Tn3-HoHo1 (vir::IacZ fusion, Cbr) pLAFRl (15), chvE::TnS from A1068 (Kmr, Tcr) pVK102 (26), chvE region from A136 (Kmr) pVK102 (26), chvE region from A136 (Tcr)
Reference
17 17 17
17
11 17; this work 17; this work 17; this work This work This work This work
45
54, 55 54, 55
45
54, 55
This work This work
1815
pMWH112, and the DNA fragment flanking the TnS insertion site of A1068 as probes. Plasmid pMWH146 was mutagenized by Tn3::HoHol insertions as described previously (42). Tn3::HoHol insertions were mapped and oriented by digestion with BamHI and EcoRI and by Southern analysis, as described previously (11). Protein analysis and immunoblotting. After 24 h of induction with 100 ,uM acetosyringone, bacteria were harvested and lysed with a French press (10,000 lb/in2). Lysates were centrifuged at low speed to remove cellular debris, and the concentration of protein in the supernatants was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining (27). Equal amounts of total protein were then electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose as described by Burnette (2). VirD2 protein was detected with polyclonal antiserum against VirD2 (39) and visualized by immunostaining with reagents purchased from Bio-Rad Laboratories. Sequence analysis. The 3.7-kb EcoRI fragment from pMWH146 was cloned into the sequencing plasmid pTZ18R (U.S. Biochemical Corp.). Nesting exonuclease III deletions (23) were generated by using materials and protocols from Promega Biotec. Chain termination sequencing was carried out by using materials and protocols from U.S. Biochemical Corp. Both strands were sequenced by the dGTP and dITP methods specified by U.S. Biochemical Corp. (48). The sequence was assembled and analyzed by using DNASTAR and GENEPRO software. Homology searches were carried out by using the FASTA algorithm (37). The cloned TnS insertion from strain A1068 was located by sequencing the surrounding region, as described previously (55).
This work
a Abbreviations: Cbr, carbenicillin resistance; Kmr, kanamycin resistance; Nair, nalidixic acid resistance; Rifr, rifampin resistance; Tcr, tetracycline resistance.
pLAFR1 (15). Ligated molecules were packaged in vitro into bacteriophage lambda particles (32) and used to transduce E. coli DHSa. Bacteria were plated on L agar supplemented with tetracycline and kanamycin to select bacteria harboring recombinant cosmids containing a Tn5 insertion and the surrounding chromosomal regions. This procedure was used to isolate plasmid pMWH102. The wild-type target sequence was isolated by colony hybridization, as follows. The DNA fragment containing the TnS insertion from pMWH102 was labeled with 32P and used to probe a library of A. tumefaciens A136 DNA cloned in the cosmid vector pVK102 (3). A cosmid clone which hybridized to this probe, pMWH112, was transferred by triparental mating (9) into A. tumefaciens chromosomal host range mutants A1008, A1059, and A1068. This clone complemented all three mutants. The 7.3-kb HindIlI fragment of pMWH112 was then cloned into the HindIII site of pVK102. The resulting plasmid, pMWH146, also complemented all three mutants. Properties of pMWH102, pMWH112, and pMWH146 are summarized in Table 1. Transposon mapping and site-specific mutagenesis. The Tn5 insertion in A1068 was mapped by digestion of pMWH102 with BgIII, HindIII, EcoRI, and Sall followed by agarose gel electrophoresis. TnS insertions in A1008 and A1059 were mapped by digestion of genomic DNA with EcoRI and HindIII and Southern hybridization (32) with ColEl::TnS,
RESULTS Initial description of chromosomal host range mutants. Three independently isolated A. tumefaciens mutants (A1008, A1059, and A1068) with chromosomal TnS insertions were previously found to have a very limited host range (17). We extended these studies by inoculating the three mutants onto Kalanchoe leaves, pea hypocotyls, sunflower stems, zinnia stems, tomato stems, Nicotiana glauca stems, and N. glauca leaf disks. None of the mutants formed tumors on Kalanchoe leaves, pea hypocotyls, tomato stems, or N. glauca leaf disks. They all induced tumors on sunflower stems, zinnia stems, and N. glauca stems, although the appearance of tumors was delayed for 2 to 3 days. The parent strain, A723, formed tumors on all of these plants, and the chvB mutant strain A1038 (11) did not form tumors on any of them. The present results confirmed the previous report, except for results with mutant A1008, which was reported to be virulent on tomato (17). This discrepancy might have resulted from differences in the age of the tomato plants or in the number of bacteria used in the assays. We confirmed the previous report (17) that the TnS insertions in these mutants were located in the chromosome by using an Eckhardt gel (13) followed by Southern hybridization with ColEl::Tn5 as probe (data not shown). The three mutants grew normally on all media, but old colonies on AB minimal agar plates appeared slightly rougher than wild-type colonies (data not shown). The mutants attached normally to representative resistant (pea and tomato) and susceptible (zinnia and sunflower) plant cells in the attachment assays of both Douglas et al. (10) and Hawes (21). vir gene induction. Since the mutants were normal in
1816
J. BACTERIOL.
HUANG ET AL.
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FIG. 1. Transcriptional induction of virB::IacZ and virE::IacZ fusions. Bacteria containing pSM243cd (virB::lacZ) or pSM358cd (virE::lacZ) were incubated in MS medium with 100 ,uM acetosyringone. At the indicated times, samples were taken and ,-galactosidase activities were determined. (A) Induction of virB; (B) induction of virE.
attachment, we analyzed another early step in tumor formation, i.e., the transcriptional inductions of the vir genes. To do this, we introduced plasmids containing vir: :lacZ fusions (Table 1) into mutant and wild-type strains by triparental mating (9). p-Galactosidase activity was measured after the mutants had been cultured in MS medium containing 100 jig of acetosyringone per ml (42). Time course experiments (Fig. 1) showed that derepression of virB and virE was greatly reduced relative to the wild-type parent strain, A723. We also measured the induction of virC, virD, and virG 24 h after the addition of acetosyringone. Induction of all of these genes was also reduced significantly in the mutants relative to wild-type cells (data not shown). Genetic analysis of strain A1068. The TnS insertions in the three strains were located as described in Materials and Methods. They map within 0.2 kb of each other (Fig. 2). We also verified by the following procedure that the TnS insertion in the mutant A1068 caused the altered virulence and vir gene induction phenotypes. Chromosomal DNA with this TnS insertion was cloned as described in Materials and Methods. The mutated region was recombined into two wild-type Agrobacterium strains, A348 and A723, by marker exchange (40). Twelve randomly chosen marker exchange mutants were avirulent on Kalanchoe leaves but virulent on sunflower stems. Induction of a virB: :lacZ fusion introduced into two of the TnS-marker exchange mutants, A723MX1 and A348MX1, was greatly reduced relative to the wild type (data not shown). Organization of the chromosomal virulence locus. To analyze the genetic and transcriptional organization of this chromosomal region, we mutagenized the 7.3-kb HindIII fragment of pMWH146 with Tn3::HoHol (42). Nineteen Tn3: :HoHol insertions were located and oriented by restriction analysis (Fig. 2). The ability of the mutagenized plasmids to complement the virulence phenotype of mutant strain A1068 was determined after the plasmids had been introduced into this strain by triparental mating. Only four
plasmids, each carrying Tn3-HoHol insertions clustered in a 1-kb region, did not complement the mutation (Fig. 2). The direction of transcription of target genes was determined by analyzing ,-galactosidase activity transcribed from lacZ fusions resulting from Tn3::HoHol insertions (42). Figure 2 shows the 3-galactosidase activity in strains carrying each Tn3::HoHol insertion and the orientation of each insertion. One insertion, 503, resulted in avirulence and expressed strong 3-galactosidase activity. Presumably, a gene involved in virulence is transcribed in the direction in which this insertion is oriented. Expression of this gene was independent of acetosyringone. Tn3::HoHol insertion 557 was recombined into the chromosome of A348 by marker exchange. The resulting mutant, MX557, did not form tumors on Kalanchoe leaves. vir gene induction in this mutant was tested by immunoblotting, to avoid the problem of background ,-galactosidase activity from the Tn3: :HoHol insertion in the chromosome. Proteins from induced and uninduced strains A348 and MX557 were separated in a sodium dodecyl sulfate-10% acrylamide gel and immunoblotted by using antiserum against virD2 (Fig. 3). The results show that induction of virD2 in this recombinant mutant is repressed, confirming at the level of protein synthesis that insertions in this region result in the observed phenotypes. Effects of inducer concentration, phosphate concentration, and pH on induction. The effects of various concentrations of acetosyringone (0, 10, 50, 100, and 200 ,uM) on induction of a virB: :lacZ fusion in wild-type strain A348 and TnS marker exchange strain A348MX1 were determined. virB induction could be detected in A348 at acetosyringone concentrations as low as 10 ,uM (Table 2). However, in A348MX1, no induction could be detected at 10 or 50 ,uM acetosyringone. When the inducer concentration was increased to 100 and 200 ,uM, induction could be detected in this mutant, although it reached a much lower level than in the wild-type strain. Expression of virG in mutants A1008, A1059, and A1068 at
AGROBACTERIUM CHROMOSOMAL VIRULENCE GENE
VOL. 172, 1990
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JOURNAL OF BACTERIOLOGY, Apr. 1990, p. 1814-1822 0021-9193/90/041814-09$02.00/0 Copyright C) 1990, American Society for Microbiology
A Chromosomal Agrobacterium tumefaciens Gene Required for Effective Plant Signal Transduction MEEI-LI WU HUANG,lt GERARD A. CANGELOSI,2 WALTER HALPERIN,1 AND EUGENE W. NESTER2* Department of Botany1 and Department of Microbiology,2 University of Washington, Seattle, Washington 98195 Received 5 October 1989/Accepted 29 December 1989 The vir gene products of Agrobactenium tumefaciens carry out the transfer of T-DNA to the plant genome. Effective transcriptional induction of the vir genes by plant signal molecules is controlled by two vir gene products, VirA and VirG. In this study we have identified and cloned a chromosomal region which is also required for vir gene induction. Transposon insertions within this region reduce induction significantly and strongly attenuate virulence, resulting in a restricted host range for infection. The reduction in vir gene transcription can be partially overcome by high concentrations of the inducer molecule acetosyringone. Expression of virG at low pH and low phosphate concentrations, which is independent of plant signals, is not affected by these mutations. Sequence analysis of the region revealed two divergent open reading frames, which we have designated chvE and ORF1. Several transposon insertions mapped in chvE; this resulted in attenuated virulence. chvE codes for a putative protein which is homologous to two periplasmic receptor proteins involved in chemotaxis and uptake of sugars. Whether ORF1 is required for virulence is uncertain. One transposon insertion resulting in avirulence maps in or near the 5' end of ORF1, and several which do not affect virulence map in its 3' end. ORF1 codes for a putative protein which is homologous to a family of transcriptional activator proteins.
The host range for Agrobacterium infection is determined largely by the T-DNA and vir regions of the Ti plasmid (28, 29, 35, 50). Increasing the expression of certain vir genes increases the virulence and host range of the bacteria (25). However, several observations have indicated that chromosomal genes are also involved in determining host range (17, 20). In this study, we found that chromosomal host range mutants originally isolated by Garfinkel and Nester (17) are defective in vir gene induction.
Strains of Agrobacterium tumefaciens containing a Ti plasmid can incite crown gall tumors on most dicotyledonous and some monocotyledonous plants. Two regions of the Ti plasmid, the transferred DNA (T-DNA) and virulence (vir) regions, are necessary for tumor formation. During infection of wounded plant tissue, the T-DNA is transferred and integrated into the plant nuclear DNA (6, 49), where its expression results in crown gall tumor formation (12, 18, 53). In addition to the Ti plasmid virulence genes, chromosomal genes chvA, chvB, and exoC are required for complete virulence (3, 11). The functioning of these genes is required for attachment of the bacteria to plant cells. Transcription of the vir genes is induced by phenolic compounds, such as acetosyringone, synthesized by wounded plant cells (43, 44). The induction system is controlled by two of the vir genes, virA and virG (45, 54). virA codes for an inner membrane protein believed to detect the signal molecules, and virG codes for a cytoplasmic protein believed to be a transcriptional activator. Mutations in either virA or virG abolish both vir gene induction and virulence. Two chromosomal genes, ros and chvD, are also somehow involved in regulating vir gene expression. In ros mutants, expression of virC and virD genes is elevated even in the absence of inducers and polysaccharide synthesis is strongly repressed. However, the induction of all other vir genes is normal, and virulence is unaffected (7). In the absence of plant inducers, the expression of virG in wild-type strains is elevated by growth under acidic conditions and by phosphate starvation. This induction is influenced by chvD (55). Mutations in chvD attenuate both vir gene induction and virulence. How these two chromosomal loci influence vir gene expression is not clear. *
MATERIALS AND METHODS
Bacterial strains, plasmids, and media. Escherichia coli DH5(x, HB101 (1), and SF800 (22) were grown in LB liquid medium and on L agar (33), supplemented when appropriate with 100 ,ug of kanamycin per ml, 100 ,ug of carbenicillin per ml, 20 ,ug of tetracycline per ml, 50 ,ug of nalidixic acid per ml, or 100 ,ug of chloramphenicol per ml. A. tumefaciens strains (Table 1) were maintained on AB minimal medium and MG/L medium (5) supplemented when appropriate with 100 p,g of kanamycin per ml, 100 ,ug of carbenicillin per ml, or S pug of tetracycline per ml. vir gene induction assays. Plasmids carrying vir::lacZ fusions (Table 1) were introduced into the appropriate A. tumefaciens strains by triparental mating (9). Cells were cultured in either MS medium (34) or induction medium (55). When appropriate, acetosyringone (Aldrich Chemical Co., Inc.) was added to the concentrations indicated in the Results. At the times indicated, 1-ml samples were removed and frozen at -70°C. After the last samples were collected, all samples were thawed and 3-galactosidase activity was assayed as described by Stachel et al. (42). Molecular cloning. Total DNA from A1068 (Table 1) was partially digested with EcoRI and electrophoresed in an agarose gel. DNA of approximately 20 kilobases (kb) was electroeluted from the gel and ligated into the EcoRI site of
Corresponding author.
t Present address: Department of Genetics, University of Washington, Seattle, WA 98195. 1814
VOL. 172, 1990
AGROBACTERIUM CHROMOSOMAL VIRULENCE GENE
TABLE 1. Agrobacterium strains and plasmids used in this study Strain or plasmid
Strains A136 A348 A722
A723
A1038 A1008 A1059 A1068 A348MX1 A723MX1 MX557
Plasmids pSM243cd
pSW161 pSW162 pSM358cd
pSW174 pMWH102
pMWH112 pMWH146
Relevant genotype or phenotype'
C58 chromosome (Rif' Nalr), no Ti plasmid A136 with pTiA6NC A114 (C58 chromosome) with pTiA6NC A114 (C58 chromosome) with pTiB6806 A723, chvB::Tn5 (Kmr) A722, chvE::TnS (Kmr) A723, chvE::Tn5 (Kmr) A723, chvE::Tn5 (Kmr) A348, chvE::Tn5 (Kmr) A723, chvE::TnS (Kmr) A348, chvE::Tn3-HoHol (CbY) or ORF1::Tn3-HoHol (Cb')
virB::Tn3-HoHol (vir::lacZ fusion, Cbr) virC::Tn3-HoHol (vir::IacZ fusion, Cb`) virD::Tn3-HoHol (vir::IacZ fusion, Cb') virE::Tn3-HoHol (vir::lacZ fusion, Cbr) virG::Tn3-HoHo1 (vir::IacZ fusion, Cbr) pLAFRl (15), chvE::TnS from A1068 (Kmr, Tcr) pVK102 (26), chvE region from A136 (Kmr) pVK102 (26), chvE region from A136 (Tcr)
Reference
17 17 17
17
11 17; this work 17; this work 17; this work This work This work This work
45
54, 55 54, 55
45
54, 55
This work This work
1815
pMWH112, and the DNA fragment flanking the TnS insertion site of A1068 as probes. Plasmid pMWH146 was mutagenized by Tn3::HoHol insertions as described previously (42). Tn3::HoHol insertions were mapped and oriented by digestion with BamHI and EcoRI and by Southern analysis, as described previously (11). Protein analysis and immunoblotting. After 24 h of induction with 100 ,uM acetosyringone, bacteria were harvested and lysed with a French press (10,000 lb/in2). Lysates were centrifuged at low speed to remove cellular debris, and the concentration of protein in the supernatants was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining (27). Equal amounts of total protein were then electrophoresed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose as described by Burnette (2). VirD2 protein was detected with polyclonal antiserum against VirD2 (39) and visualized by immunostaining with reagents purchased from Bio-Rad Laboratories. Sequence analysis. The 3.7-kb EcoRI fragment from pMWH146 was cloned into the sequencing plasmid pTZ18R (U.S. Biochemical Corp.). Nesting exonuclease III deletions (23) were generated by using materials and protocols from Promega Biotec. Chain termination sequencing was carried out by using materials and protocols from U.S. Biochemical Corp. Both strands were sequenced by the dGTP and dITP methods specified by U.S. Biochemical Corp. (48). The sequence was assembled and analyzed by using DNASTAR and GENEPRO software. Homology searches were carried out by using the FASTA algorithm (37). The cloned TnS insertion from strain A1068 was located by sequencing the surrounding region, as described previously (55).
This work
a Abbreviations: Cbr, carbenicillin resistance; Kmr, kanamycin resistance; Nair, nalidixic acid resistance; Rifr, rifampin resistance; Tcr, tetracycline resistance.
pLAFR1 (15). Ligated molecules were packaged in vitro into bacteriophage lambda particles (32) and used to transduce E. coli DHSa. Bacteria were plated on L agar supplemented with tetracycline and kanamycin to select bacteria harboring recombinant cosmids containing a Tn5 insertion and the surrounding chromosomal regions. This procedure was used to isolate plasmid pMWH102. The wild-type target sequence was isolated by colony hybridization, as follows. The DNA fragment containing the TnS insertion from pMWH102 was labeled with 32P and used to probe a library of A. tumefaciens A136 DNA cloned in the cosmid vector pVK102 (3). A cosmid clone which hybridized to this probe, pMWH112, was transferred by triparental mating (9) into A. tumefaciens chromosomal host range mutants A1008, A1059, and A1068. This clone complemented all three mutants. The 7.3-kb HindIlI fragment of pMWH112 was then cloned into the HindIII site of pVK102. The resulting plasmid, pMWH146, also complemented all three mutants. Properties of pMWH102, pMWH112, and pMWH146 are summarized in Table 1. Transposon mapping and site-specific mutagenesis. The Tn5 insertion in A1068 was mapped by digestion of pMWH102 with BgIII, HindIII, EcoRI, and Sall followed by agarose gel electrophoresis. TnS insertions in A1008 and A1059 were mapped by digestion of genomic DNA with EcoRI and HindIII and Southern hybridization (32) with ColEl::TnS,
RESULTS Initial description of chromosomal host range mutants. Three independently isolated A. tumefaciens mutants (A1008, A1059, and A1068) with chromosomal TnS insertions were previously found to have a very limited host range (17). We extended these studies by inoculating the three mutants onto Kalanchoe leaves, pea hypocotyls, sunflower stems, zinnia stems, tomato stems, Nicotiana glauca stems, and N. glauca leaf disks. None of the mutants formed tumors on Kalanchoe leaves, pea hypocotyls, tomato stems, or N. glauca leaf disks. They all induced tumors on sunflower stems, zinnia stems, and N. glauca stems, although the appearance of tumors was delayed for 2 to 3 days. The parent strain, A723, formed tumors on all of these plants, and the chvB mutant strain A1038 (11) did not form tumors on any of them. The present results confirmed the previous report, except for results with mutant A1008, which was reported to be virulent on tomato (17). This discrepancy might have resulted from differences in the age of the tomato plants or in the number of bacteria used in the assays. We confirmed the previous report (17) that the TnS insertions in these mutants were located in the chromosome by using an Eckhardt gel (13) followed by Southern hybridization with ColEl::Tn5 as probe (data not shown). The three mutants grew normally on all media, but old colonies on AB minimal agar plates appeared slightly rougher than wild-type colonies (data not shown). The mutants attached normally to representative resistant (pea and tomato) and susceptible (zinnia and sunflower) plant cells in the attachment assays of both Douglas et al. (10) and Hawes (21). vir gene induction. Since the mutants were normal in
1816
J. BACTERIOL.
HUANG ET AL.
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HOURS
FIG. 1. Transcriptional induction of virB::IacZ and virE::IacZ fusions. Bacteria containing pSM243cd (virB::lacZ) or pSM358cd (virE::lacZ) were incubated in MS medium with 100 ,uM acetosyringone. At the indicated times, samples were taken and ,-galactosidase activities were determined. (A) Induction of virB; (B) induction of virE.
attachment, we analyzed another early step in tumor formation, i.e., the transcriptional inductions of the vir genes. To do this, we introduced plasmids containing vir: :lacZ fusions (Table 1) into mutant and wild-type strains by triparental mating (9). p-Galactosidase activity was measured after the mutants had been cultured in MS medium containing 100 jig of acetosyringone per ml (42). Time course experiments (Fig. 1) showed that derepression of virB and virE was greatly reduced relative to the wild-type parent strain, A723. We also measured the induction of virC, virD, and virG 24 h after the addition of acetosyringone. Induction of all of these genes was also reduced significantly in the mutants relative to wild-type cells (data not shown). Genetic analysis of strain A1068. The TnS insertions in the three strains were located as described in Materials and Methods. They map within 0.2 kb of each other (Fig. 2). We also verified by the following procedure that the TnS insertion in the mutant A1068 caused the altered virulence and vir gene induction phenotypes. Chromosomal DNA with this TnS insertion was cloned as described in Materials and Methods. The mutated region was recombined into two wild-type Agrobacterium strains, A348 and A723, by marker exchange (40). Twelve randomly chosen marker exchange mutants were avirulent on Kalanchoe leaves but virulent on sunflower stems. Induction of a virB: :lacZ fusion introduced into two of the TnS-marker exchange mutants, A723MX1 and A348MX1, was greatly reduced relative to the wild type (data not shown). Organization of the chromosomal virulence locus. To analyze the genetic and transcriptional organization of this chromosomal region, we mutagenized the 7.3-kb HindIII fragment of pMWH146 with Tn3::HoHol (42). Nineteen Tn3: :HoHol insertions were located and oriented by restriction analysis (Fig. 2). The ability of the mutagenized plasmids to complement the virulence phenotype of mutant strain A1068 was determined after the plasmids had been introduced into this strain by triparental mating. Only four
plasmids, each carrying Tn3-HoHol insertions clustered in a 1-kb region, did not complement the mutation (Fig. 2). The direction of transcription of target genes was determined by analyzing ,-galactosidase activity transcribed from lacZ fusions resulting from Tn3::HoHol insertions (42). Figure 2 shows the 3-galactosidase activity in strains carrying each Tn3::HoHol insertion and the orientation of each insertion. One insertion, 503, resulted in avirulence and expressed strong 3-galactosidase activity. Presumably, a gene involved in virulence is transcribed in the direction in which this insertion is oriented. Expression of this gene was independent of acetosyringone. Tn3::HoHol insertion 557 was recombined into the chromosome of A348 by marker exchange. The resulting mutant, MX557, did not form tumors on Kalanchoe leaves. vir gene induction in this mutant was tested by immunoblotting, to avoid the problem of background ,-galactosidase activity from the Tn3: :HoHol insertion in the chromosome. Proteins from induced and uninduced strains A348 and MX557 were separated in a sodium dodecyl sulfate-10% acrylamide gel and immunoblotted by using antiserum against virD2 (Fig. 3). The results show that induction of virD2 in this recombinant mutant is repressed, confirming at the level of protein synthesis that insertions in this region result in the observed phenotypes. Effects of inducer concentration, phosphate concentration, and pH on induction. The effects of various concentrations of acetosyringone (0, 10, 50, 100, and 200 ,uM) on induction of a virB: :lacZ fusion in wild-type strain A348 and TnS marker exchange strain A348MX1 were determined. virB induction could be detected in A348 at acetosyringone concentrations as low as 10 ,uM (Table 2). However, in A348MX1, no induction could be detected at 10 or 50 ,uM acetosyringone. When the inducer concentration was increased to 100 and 200 ,uM, induction could be detected in this mutant, although it reached a much lower level than in the wild-type strain. Expression of virG in mutants A1008, A1059, and A1068 at
AGROBACTERIUM CHROMOSOMAL VIRULENCE GENE
VOL. 172, 1990
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