Polygalacturonase Production by Agrobacterium tumefaciens Biovar 3

Agrobacterium tumefaciens biovar 3 causes both crown gall and root decay of grape. Twenty-two Agrobacterium strains representing biovars 1, 2, and 3 were analyzed for tumorigenicity, presence of a Ti plasmid, ability to cause grape seedling root decay, and pectolytic activity. All of the biovar 3 strains, regardless of their tumorigenicity or presence of a Ti plasmid, caused root decay and were pectolytic, whereas none of the biovar 1 and 2 strains had these capacities. Isoelectrically focused gels that were activity stained with differentially buffered polygalacturonate-agarose overlays revealed that all of the biovar 3 strains produced a single polygalacturonase with a pH optimum of 4.5 and pIs ranging from 4.8 to 5.2. The enzyme was largely extracellular and was produced constitutively in basal medium supplemented with a variety of carbon sources including polygalacturonic acid. Lesions on grape seedling roots inoculated with A. tumefaciens biovar 3 strain CG49 yielded polygalacturonase activity with a pI similar to that of the enzyme produced by the bacterium in culture. These observations support the hypothesis that the polygalacturonase produced by A. tumefaciens biovar 3 has a role in grape root decay.     

Attachment, Chemotaxis, and Multiplication of Agrobacterium tumefaciens Biovar 1 and Biovar 3 on Grapevine and Pea

Tumorigenic (CG49) and nontumorigenic (CG484) strains of Agrobacterium tumefaciens bv. 3 attached to grape roots at a higher level than did a nonpectinolytic mutant of CG49 (CG50) or a tumorigenic strain of A. tumefaciens bv. 1 (CG628). Strains attached equally well to wounded and unwounded grape roots. Strains responded differently to pea plants in that biovar 3 strains consistently attached to unwounded roots at a lower level than they did to wounded roots, whereas CG628 attached equally well regardless of wounding. The lowest levels of attachment to pea roots were consistently observed for CG50. Population curves were calculated for the strains inoculated into wound sites on grape and pea roots. A. tumefaciens bv. 3 wild-type strains developed greater populations at wound sites on grape roots after 100 h (resulting in root decay) than did CG50 or CG628. Population curves for strains at wound sites on pea roots were different from those on grape roots. There were no significant differences in populations after 100 h, and no strains caused root decay. No differences in the chemotaxis of wild-type and mutant A. tumefaciens bv. 3 strains towards grape roots, crown pieces, or root extracts were observed, but the biovar 1 strain, CG628, always migrated the greatest distance towards all substrates. Polygalacturonase production may affect attachment to grape roots and multiplication of A. tumefaciens bv. 3 at wound sites and thus be associated with the specificity of the bacterium for grape.     

Characterization of Tn904 insertions in octopine Ti plasmid mutants of Agrobacterium tumefaciens.

Seven Tn904 insertion mutants of pTi Ach5 affecting Agrobacterium tumefaciens virulence were studied. The mutant character was shown to be plasmid borne. Four of these mutants were avirulent and carried an insertion in restriction endonuclease HpaI fragment 12, a 3.3-megadalton fragment, which therefore appears to be a Ti plasmid region essential for virulence. Two mutants were attenuated in virulence. The inserts mapped close to HpaI fragment 12. One mutant giving rise to small tumors with excessive adventitious root formation on Kalanchoe daigremontiana carried an insertion in the right side of the common sequence in the deoxyribonucleic acid of the Ti plasmid detected in crown gall tumors. The insertion behavior of Tn904 was studied by analyzing 11 independently isolated and randomly chosen mutants. The Tn904 inserts did not affect oncogenicity, tumor morphology, bacterial transfer functions, octopine catabolism functions, or vital parts of the Ti plasmid, such as the origin of replication. Most of the Tn904 inserts were concentrated in a small part of the map. The size of additional deoxyribonucleic acid as a result of Tn904 inserts varied between 5 and 15 megadaltons. In two cases a Ti plasmid was found with two Tn904 insertions at different positions.     

Deletion derivatives of pAgK84 and their use in the analysis of Agrobacterium plasmid functions.

The 47.7-kb plasmid pAgK84, present in Agrobacterium radiobacter strain K84, confers production of a novel, highly specific, antiagrobacterial antibiotic called agrocin 84. Strain K84 is used commercially to biocontrol crown gall caused by agrocin 84-susceptible strains of Agrobacterium tumefaciens. Efficient biocontrol is dependent upon production of agrocin 84 by strain K84. Starting with a derivative of pAgK84 containing a Tn5 insertion, a series of deletion derivatives of the plasmid were isolated. The smallest of these, pJS500, contains about 8 kb of the original agrocin plasmid and localized the replication functions to between 4 and 6 o'clock on the physical map. A smaller derivative, produced by clonal rescue of a Tn5 insertion in the 4 o'clock region, further localized the minimal replication functions to a 1.5-kb region mapping between coordinates 18.1 and 19.6. Analysis of plasmid stability indicated that functions required for maintenance of the plasmid under nonselective conditions are tightly linked to the minimal replication region. This region also encodes incompatibility functions; the deletion derivatives were all incompatible with the wild-type pAgK84. The stability/replication locus of pAgK84 maps just anticlockwise from the Tra region. This region is retained fully in pAgK1026, the directed Tra- derivative of pAgK84 which is now in use as the primary crown gall biocontrol agent in Australia. One of the deletion derivatives, the 15-kb pJS400, was used as a vector to clone the KpnI fragments of an octopine-type Ti plasmid. Traits known to be encoded on these fragments were expressed and properly regulated in Agrobacterium hosts. One clone, encoding the Ti plasmid replication/incompatibility region, was used to cure IncRh1 Ti plasmids from their hosts. This clone also was found to be incompatible with pAtK84b, a large plasmid encoding opine catabolism present in A. radiobacter strain K84. This indicates that the opine catabolic plasmid is closely related to the IncRh1 Ti plasmids.     

Highly tumorigenic Agrobacterium tumefaciens strain from crown gall tumors of chrysanthemum

A wild-type Agrobacterium tumefaciens strain CNI5 isolated from crown gall of chrysanthemum (Dendranthema grandiflora Tzvelev) was characterized. Strain CNI5 was classified into biovar 1, based on physiological and biochemical characteristics, and was resistant to ampicillin. Strain CNI5 induced tumors at a higher frequency and on a larger area of explants in most tested plant species, especially in chrysanthemum cultivars, than the octopine-type strain C58C1cmr (pTiB6S3). Agropine and mannopine were detected in tumors induced by strain CNI5 and were specifically catabolized by this strain. Strain CNI5 harbored five plasmids including one plasmid that shared sequence similarity to TL-DNA of the octopine-type Ti plasmid and four cryptic plasmids.     

The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity.

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by Tn5 mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T7 phi 10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.     

Expression of a crown gall biological control phenotype in an avirulent strain of Agrobacterium vitis by addition of the trifolitoxin production and resistance genes

Background

Agrobacterium vitis is a causal agent of crown-gall disease. Trifolitoxin (TFX) is a peptide antibiotic active only against members of a specific group of α-proteobacteria that includes Agrobacterium and its close relatives. The ability of TFX production by an avirulent strain of Agrobacterium to reduce crown gall disease is examined here.

Results

TFX was shown to be inhibitory in vitro against several A. vitis strains. TFX production, expressed from the stable plasmid pT2TFXK, conferred biological control activity to an avirulent strain of A. vitis. F2/5, against three virulent, TFX-sensitive strains of A. vitis tested on Nicotiana glauca. F2/5(pT2TFXK) is significantly reduces number and size of galls when co-inoculated with tumorigenic strain CG78 at a 10:1 ratio, but is ineffective at 1:1 or 1:10 ratios. F2/5(pT2TFXK) is effective when co-inoculated with tumorigenic strain CG435 at 10:1 and 1:1 ratios, but not at a 1:10 ratio. When F2/5(pT2TFXK) is co-inoculated with CG49 at a 10:1 ratio, the incidence of gall formation does not decline but gall size decreases by more than 70%. A 24 h pre-inoculation with F2/5(pT2TFXK) does not improve biological control at the 1:10 ratio.

Conclusions

TFX production by an avirulent strain of Agrobacterium does confer in that strain the ability to control crown gall disease on Nicotiana glauca. This is the first demonstration that the production of a ribosomally synthesized, post-translationally modified peptide antibiotic can confer reduction in plant disease incidence from a bacterial pathogen.     

Biological Control of Agrobacterium tumefaciens, Colonization, and pAgK84 Transfer with Agrobacterium radiobacter K84 and the Tra Mutant Strain K1026

The efficacies of Agrobacterium radiobacter K84 and K1026 in root colonization, crown gall control, and plasmid transfer were compared. Levels of root colonization by K84 and K1026 of Montclar and Nemaguard peach seedlings were similar during the 21 days of the experiment. Four strains of A. tumefaciens bv. 1 were used for soil inoculations in biological control experiments on GF677 and Adafuel peach x almond rootstocks; two were sensitive and two were resistant to agrocin 84. Both strains K84 and K1026 were very efficient in controlling the sensitive strains, but some tumors appeared with both treatments. In the biocontrol of resistant strains, no galls were observed in K1026-treated plants, but some K84-treated plants had galls. Recovery of agrobacteria from galls in experiments with sensitive and resistant strains showed that all of the isolates from the controls or K1026-treated plants and most of the isolates from K84-treated plants had the same characteristics as the inoculated strains. Nine isolates from the K84-treated plants growing in soil inoculated with one resistant strain were virulent and produced agrocin 84. These isolates had a plasmid that hybridized with a probe prepared with the BamHI C fragment from pAgK84. These results show the efficiency of K1026 in biocontrol of agrocin 84-sensitive and -resistant strains of A. tumefaciens and suggest the use of K1026 as a safer organism than K84 for biological control of crown gall.     

Biological Control of Crown Gall in Grapevine and Raspberry by Two Pseudomonas spp. with a Wide Spectrum of Antagonistic Activity

Pseudomonas aureofaciens B-4117 and P. fluorescens CR330D inhibited the growth of a wide range of plant pathogens, including Agrobacterium tumefaciens , when tested on agar media. In a series of nursery-based trials with natural pathogen inoculum, application of either B-4117 or CR330D significantly reduced the incidence and severity of crown gall caused by A. tumefaciens on grapevine and raspberry. The extent of disease control depended upon the variety tested. Both bacteria reduced disease during seedling root production and grafting. The disease incidence on root cuttings of three grapevine varieties was reduced by 56-80% and the disease severity index (DSI) was decreased by 75-86%. Depending on the scion variety, the number of healthy rooted grafts increased by 2-3.5-fold, while the DSI was reduced by 1.5-3-fold. The results suggest that there is potential in using these antagonists to diminish the influence of latent rootstock infection on graft sensitivity to crown gall. Pretreatment of rooted raspberry seedlings with P. aureofaciens B-4117 prevented the development of crown galls caused by A. tumefaciens strain K24 or by a mixture of A. tumefaciens pathogenic strains previously isolated from raspberry. Both Pseudomonas spp. persisted on the root surfaces of inoculated vine cuttings and in non-sterile soil. The advantages of using the antagonistic bacteria as biocontrol agents of crown gall are discussed.     

A truncated Tn916-like element in a clinical isolate of Enterococcus faecium.

A 58.7-kb nonconjugative plasmid (pKQ1) previously reported in a clinical isolate of Enterococcus faecium was found to contain both a tetM and an erythromycin resistance (erm) determinant. The plasmid contained a region homologous to the A, F, H, and G HincII fragments of Tn916. However, the 4.8-kb B fragment of Tn916 which contained the tetM determinant was replaced by a 7.3-kb fragment, and the 3.6-kb HincII C fragment of Tn916 was missing. An element homologous to Tn917 was juxtaposed to the truncated Tn916-like element. The Tn917-like element was similar in size to the erm transposon Tn917 as determined by a ClaI restriction digest which spanned approximately 99% of the transposon. When Bacillus subtilis or Streptococcus sanguis were transformed with pKQ1, no zygotically induced transposition of the tetM element was detected. Similarly no transposition of the Tn917-like element was detected.     

ACC deaminase activity in avirulent Agrobacterium tumefaciens D3

Some plant-growth-promoting bacteria encode the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, which breaks down ACC, the direct precursor of ethylene biosynthesis in all higher plants, into ammonia and α-ketobutyrate and, as a result, reduces stress ethylene levels in plants caused by a wide range of biotic and abiotic stresses. It was previously shown that ACC deaminase can inhibit crown gall development induced by Agrobacterium tumefaciens and can partially protect plants from this disease. Agrobacterium tumefaciens D3 has been previously reported to contain a putative ACC deaminase structural gene (acdS) and a regulatory gene (acdR = lrpL). In the present study, it was found that A. tumefaciens D3 is an avirulent strain. ACC deaminase activity and its regulation were also characterized. Under gnotobiotic conditions, wild-type A. tumefaciens D3 was shown to be able to promote plant root elongation, while the acdS and lrpL double mutant strain A. tumefaciens D3-1 lost that ability. When co-inoculated with the virulent strain, A. tumefaciens C58, in wounded castor bean plants, both the wild-type A. tumefaciens D3 and the mutant A. tumefaciens D3-1 were found to be able to significantly inhibit crown gall development induced by A. tumefaciens C58.     

Isolation and genetic analysis of an Agrobacterium tumefaciens avirulent mutant with a chromosomal mutation produced by transposon mutagenesis.

A transposon 5 (Tn5) insertion was introduced into the genome of A. tumefaciens (A-208 strain harboring a nopaline type Ti-plasmid) using a conjugative pJB4JI plasmid containing Tn5. Five thousand transconjugants were assayed for virulence on carrot (Daucus carota L.) disks; 54 isolates were avirulent or very attenuated. The cellular localization (plasmid or chromosome) of the Tn5 insertion in those isolates were identified by Southern hybridization analysis. An avirulent mutant (B-90 strain) with the Tn5 insertion in the chromosome was selected and characterized. The mutant had the same growth rate as that of the parent strain in L-broth. The mutant and the parent strain had similar attachment ability to carrot root cells. Tn5 was inserted into one site of the chromosome. The wild-type target chromosomal region (1281 base pairs) was cloned and sequenced. An open reading frame (ORF) consisting of 395 base pairs was identified. The wild-type DNA fragment (1.6 kb) containing the ORF introduced into B-90 strain complemented the avirulent phenotype of the strain. A soluble protein was predicted from the ORF. The Tn5 was inserted near the 3'-terminal of the ORF. Homology search of this ORF found no significant homology to known genes and proteins. Thus, the ORF identified in this paper seems to be a new chromosomal virulence gene of A. tumefaciens.     

Hairy-root-inducing plasmid: physical map and homology to tumor-inducing plasmids.

A physical map was constructed for the 250-kilobase plasmid pRiA4b, which confers the virulence properties of a strain of Agrobacterium rhizogenes for hairy root disease in plants. The complete HindIII and KpnI restriction map was determined from a collection of overlapping HindIII partial digest clones. Homologous regions with two well-characterized plasmids that confer virulence for crown gall disease, plasmids pTiA6 and pTiT37, were mapped on pRiA4b. As much as 160 kilobases of pRiA4b had detectable homology to one or both of these crown-gall-tumor-inducing plasmids. About 33 kilobases of pRiA4b hybridized to the vir region of pTiA6, a segment of DNA required for virulence of Agrobacterium tumefaciens. Portions of pTiA6 and pTiT37 transferred into plant cells in crown gall disease (T-DNA), shared limited homology with scattered regions of pRiA4b. The tumor morphology loci tms-1 and tms-2 from the T-DNA of pTiA6 hybridized to pRiA4b. A T-DNA fragment containing the tml and tmr tumor morphology loci also hybridized to pRiA4b, but the homology has not been defined to a locus and is probably not specific to tmr. A segment of pRiA4b T-DNA which was transferred into plant cells in hairy root disease lacked detectable homology to pTiA6 and had limited homology at one end to the T-DNA of pTiT37.     

Requirement for chemotaxis in pathogenicity of Agrobacterium tumefaciens on roots of soil-grown pea plants.

Agrobacterium tumefaciens Tn5 mutants deficient in chemotaxis to root exudates were used to study the significance of chemotaxis in crown gall pathogenesis. Mutants deficient in motility and in chemotaxis were fully virulent when inoculated by direct immersion in inoculum, followed by growth for 2 weeks in moist growth pouches. Ability of mutant bacteria to move through soil to infect roots was tested by planting wounded seedlings into air-dried soil or sand that had been infested with inoculum. Mutant bacteria were almost as virulent as the parent on plants grown in sand but were avirulent on soil-grown plants.     

Analysis of the ruv locus of Escherichia coli K-12 and identification of the gene product.

The ruv gene of Escherichia coli, which is associated with inducible mechanisms of DNA repair and recombination, has been cloned into the low-copy plasmid vector pHSG415. The recombinant plasmid pPVA101 fully complements the DNA repair-deficient phenotype of ruv mutants. Restriction endonuclease analysis of this plasmid revealed a 10.6-kilobase (kb) HindIII DNA insert which contained a 7.7-kb PstI fragment identified as being from the chromosomal ruv region. Deletion analysis and Tn1000 insertional inactivation of ruv function located the ruv coding region to a 2.2-kb section of the cloned DNA fragment. A comparison of the proteins encoded by ruv wild-type and mutant plasmids identified the gene product as a protein of molecular weight 41,000.     

Cloning and expression of Clostridium acetobutylicum phosphotransbutyrylase and butyrate kinase genes in Escherichia coli.

A 13.6-kilobase (kb) Sau3AI restriction endonuclease fragment of Clostridium acetobutylicum DNA cloned into pBR322 enabled Escherichia coli ato mutants to grow on butyrate as a sole carbon source (But+). Complementation of the ato defect by the recombinant plasmid pJC6 was due to expression of the genes for phosphotransbutyrylase (PTB) and butyrate kinase (BK). Both genes were efficiently expressed in E. coli, as their products were readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts. PTB was found to have a polypeptide subunit molecular weight of approximately 31,000, while that of BK was approximately 39,000. Deletion analysis and Tn5 mutagenesis of plasmid pJC7 (a But+ subclone containing a 4.4-kb BamHI fragment from the insert of pJC6) localized the PTB and BK genes within a region spanning approximately 2.9 kb. Preliminary evidence suggests that the two genes may form an operon that is transcribed as a single unit from a promoter of clostridial origin within the 4.4-kb insert of pJC7.     

Identification of the product of an Agrobacterium tumefaciens chromosomal virulence gene

The chvB operon of Agrobacterium tumefaciens is required for bacterial attachment to plant cells and for efficient crown gall tumor formation. As defined by the virulence phenotypes of mutants with transposon insertions mapping in the region, the operon was previously mapped to a 5-kilobase (kb) stretch of chromosomal DNA. We report here that the operon is actually about 8.5 kb long and that it contains a 7-kb gene coding for a large membrane protein involved in the synthesis of cyclic beta-1,2-glucan. Mutants with transposon insertions within the 5-kb phenotypically defined operon do not synthesize this functional protein, do not synthesize beta-1,2-glucan, and do not form tumors. However, mutants with insertions that map up to 3.5 kb downstream of the phenotypically defined operon synthesize truncated proteins that are active in beta-1,2-glucan synthesis. These mutants form tumors. The truncated proteins correspond closely in size with the map positions of the insertions, suggesting that the insertions truncate the proteins by translational termination. A plasmid that contains only the phenotypically defined chvB operon also codes for a truncated protein. A fusion product between the protein and beta-galactosidase carried on a Tn3-HoHo1 insertion was observed in one mutant. Partial trypsin digestion of wild-type inner membranes generated truncated proteins that were active in beta-1,2-glucan synthesis, demonstrating that a large portion of the protein is not required for beta-1,2-glucan synthesis. The correlation between beta-1,2-glucan synthesis by the truncated proteins and tumorigenesis strongly implicates the polysaccharide product of this protein in tumor formation.     

Identification and characterization of plant genes involved in Agrobacterium-mediated plant transformation by virus-induced gene silencing

Genetic transformation of plant cells by Agrobacterium tumefaciens represents a unique case of trans-kingdom sex requiring the involvement of both bacterial virulence proteins and plant-encoded proteins. We have developed in planta and leaf-disk assays in Nicotiana benthamiana for identifying plant genes involved in Agrobacterium-mediated plant transformation using virus-induced gene silencing (VIGS) as a genomics tool. VIGS was used to validate the role of several genes that are either known or speculated to be involved in Agrobacterium-mediated plant transformation. We showed the involvement of a nodulin-like protein and an alpha-expansin protein (alpha-Exp) during Agrobacterium infection. Our data suggest that alpha-Exp is involved during early events of Agrobacterium-mediated transformation but not required for attaching A. tumefaciens. By employing the combination of the VIGS-mediated forward genetics approach and an in planta tumorigenesis assay, we identified 21 ACG (altered crown gall) genes that, when silenced, produced altered crown gall phenotypes upon infection with a tumorigenic strain of A. tumefaciens. One of the plant genes identified from the screening, Histone H3 (H3), was further characterized for its biological role in Agrobacterium-mediated plant transformation. We provide evidence for the role of H3 in transfer DNA integration. The data presented here suggest that the VIGS-based approach to identify and characterize plant genes involved in genetic transformation of plant cells by A. tumefaciens is simple, rapid, and robust and complements other currently used approaches.     

Host range conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens.

The host range of Agrobacterium tumefaciens 1D1109, known to induce crown gall only on grapevine (Vitis spp.), was extended to include many plant species by transferring a tumor-inducing plasmid (pTi) from strain 1D1, a broad-host-range pathogen. The pTi plasmid was mobilized by the conjugative plasmid pRK2, which was inserted into 1D1 by mating with Escherichia coli J53(pRK2). The resulting transconjugants were screened for their ability to induce crown gall tumors on hosts other than grapevine by inoculation into sunflower. Transconjugants that were virulent on sunflower were then tested on 36 different host plants and compared with host-limited strain 1D1109 and the donor strain. Two transconjugants induced tumors on the same 28 plant species as those of the original plasmid donor 1D1(pRK2) (pTi). These results show that pRK2 promoted transfer of the pTi plasmid and suggest that the pTi plasmid rather than the A. tumefaciens chromosome determined the host range of the pathogen. Insertion of pRK2 alone did not extend the host range of strain 1D1109. Insertion of pS-a into A. tumefaciens 1D1 by mating with E. coli J53-1 (pS-a) resulted in the concomitant loss of pTi and virulence. There appears to be incompatibility between pTi and pS-a.