The Effect of the Agrobacterium tumefaciens attR Mutation on Attachment and Root Colonization Differs between Legumes and Other Dicots (original) (raw)
Related papers
Journal of Bacteriology, 1997
An early step in crown gall tumor formation involves the attachment of Agrobacterium tumefaciens to host plant cells. A. tumefaciens C58::A205 (C58 attR) is a Tn3HoHo1 insertion mutant that was found to be avirulent on Bryophyllum daigremontiana and unable to attach to carrot suspension cells. The mutation mapped to an open reading frame encoding a putative protein of 247 amino acids which has significant homology to transacetylases from many bacteria. Biochemical analysis of polysaccharide extracts from wild-type strain C58 and the C58::A205 mutant showed that the latter was deficient in the production of a cell-associated polysaccharide. Anion-exchange chromatography followed by 1 H nuclear magnetic resonance and gas chromatography-mass spectrometry analyses showed that the polysaccharide produced by strain C58 was an acetylated, acidic polysaccharide and that the polysaccharide preparation contained three sugars: glucose, glucosamine, and an unidentified deoxy-sugar. Application of the polysaccharide preparation from strain C58 to carrot suspension cells prior to inoculation with the bacteria effectively inhibited attachment of the bacteria to the carrot cells, whereas an identical preparation from strain C58::A205 had no inhibitory effect and did not contain the acidic polysaccharide. Similarly, preincubation of Arabidopsis thaliana root segments with the polysaccharide prevented attachment of strain C58 to that plant. This indicates that the acidic polysaccharide may play a role in the attachment of A. tumefaciens to host soma plant cells.
Root colonization by Agrobacterium tumefaciens is reduced in cel, attB, attD, and attR mutants
Applied and Environmental Microbiology
Root colonization by Agrobacterium tumefaciens was measured by using tomato and Arabidopsis thaliana roots dipped in a bacterial suspension and planted in soil. Wild-type bacteria showed extensive growth on tomato roots; the number of bacteria increased from 10 3 bacteria/cm of root length at the time of inoculation to more than 10 7 bacteria/cm after 10 days. The numbers of cellulose-minus and nonattaching attB, attD, and attR mutant bacteria were less than 1/10,000th the number of wild-type bacteria recovered from tomato roots. On roots of A. thaliana ecotype Landsberg erecta, the numbers of wild-type bacteria increased from about 30 to 8,000 bacteria/cm of root length after 8 days. The numbers of cellulose-minus and nonattaching mutant bacteria were 1/100th to 1/10th the number of wild-type bacteria recovered after 8 days. The attachment of A. tumefaciens to cut A. thaliana roots incubated in 0.4% sucrose and observed with a light microscope was also reduced with cel and att mutants. These results suggest that cellulose synthesis and attachment genes play a role in the ability of the bacteria to colonize roots, as well as in bacterial pathogenesis.
Virulence of Agrobacterium tumefaciens Strain A281 on Legumes
Plant Physiology, 1987
This study addresses the basis of host range on legumes of Agrobacterium tumefaciens strain A281, an LL-succinamopine strain. We tested virulence of T-DNA and vir region constructs from this tumor-inducing (Ti) plasmid with complementary Ti plasmid re,gions from heterologous nopaline and octopine strains. Agrobacterium tumefaciens is the etiologic agent of crown gall. Oncogenic strains ofA. tumefaciens harbor large plasmids, called Ti2 plasmids (for review, see Ref. 17). Part of the Ti plasmidthe T-DNA-is transferred to the plant where it is stably maintained in the nuclear DNA (4, 5). The T-DNA contains genes that are transcribed in the plant (4) encoding enzymes for opine synthesis (29) and phytohormone synthesis (1, 2, 27, 30). A second region of approximately 30 to 40 kb of the Ti plasmid that is involved in tumorigenesis but is not maintained in the tumor is the virulence region. Drummond and Chilton (10) found extensive regions of DNA that are conserved on several wide host range Ti plasmids including octopine, nopaline, and L,L-succinamopine types. The vir genes and T-DNA are located in regions exhibiting this extensive homology. Many strains ofA. tumefaciens exhibit a broad host range, i.e. they incite tumors on several dicotyledonous angiosperms and on some gymnosperms (7). Some strains ofAgrobacterium incite tumors on only a limited range ofplants-mainly gragevine (26). Host range has been shown to be determined by the type of Ti plasnnid present in the bacterium (24, 31). Loci specific for hostrange have been mapped on the Ti plasmids jn the T-DNA region (3, 16) as well as outside the T-DNA (12, 21, 22). Hoekema et al. (14, 15) and de Framond et aL. (8) demonstrated that the Ti plasmid could be divided into two separately replicating plasmids in the same bacterium and induce tumors similar to wild type on the host plants tested. We report here investigations on the host range of A. tutnefaciens strain A281 using this binary system. Our purpose was to determine if virulence on legumes was primarily a property of the T-DNA or of the vir region of pTi Bo542 by testing the T-DNA and vir regions with complementary regions of Ti plasmids from other strains. We found that the interactions of the T-DNAs and vir reons 'Supported in part by Department of Energy grant DE-AC02-81ER10888 to R. N. Beachy. E. Eli. was supported by the Division of Biology and Biomedical Sciences at Washington University and by a tellowsjhip from Pioneer Hi-Bred International Inc.
Quantification of Agrobacterium tumefaciens C58 attachment to Arabidopsis thaliana roots
FEMS Microbiology Letters, 2017
Agrobacterium tumefaciens is the causal agent of crown gall disease and is a vector for DNA transfer in transgenic plants. The transformation process by A. tumefaciens has been widely studied, but the attachment stage has not been well characterized. Most measurements of attachment have used microscopy and colony counting, both of which are labor and time intensive. To reduce the time and effort required to analyze bacteria attaching to plant tissues, we developed a quantitative real-time PCR (qPCR) assay to quantify attached A. tumefaciens using the chvE gene as marker for the presence of the bacteria. The qPCR detection threshold of A. tumefaciens from pure culture was 10 4 cell equivalents/ml. The A. tumefaciens minimum threshold concentration from root-bound populations was determined to be 10 5 cell equivalents/ml inoculum to detect attachment above background. The qPCR assay can be used for measuring A. tumefaciens attachment in applications such as testing the effects of mutations on bacterial adhesion molecules or biofilm formation, comparing attachment across various plant species and ecotypes, and detecting mutations in putative attachment receptors expressed in plant roots.
Plant responses to Agrobacterium tumefaciens and crown gall development
Frontiers in Plant Science, 2014
Agrobacterium tumefaciens causes crown gall disease on various plant species by introducing its T-DNA into the genome. Therefore, Agrobacterium has been extensively studied both as a pathogen and an important biotechnological tool. The infection process involves the transfer of T-DNA and virulence proteins into the plant cell. At that time the gene expression patterns of host plants differ depending on the Agrobacterium strain, plant species and cell-type used. Later on, integration of the T-DNA into the plant host genome, expression of the encoded oncogenes, and increase in phytohormone levels induce a fundamental reprogramming of the transformed cells. This results in their proliferation and finally formation of plant tumors. The process of reprogramming is accompanied by altered gene expression, morphology and metabolism. In addition to changes in the transcriptome and metabolome, further genome-wide ("omic") approaches have recently deepened our understanding of the genetic and epigenetic basis of crown gall tumor formation. This review summarizes the current knowledge about plant responses in the course of tumor development. Special emphasis is placed on the connection between epigenetic, transcriptomic, metabolomic, and morphological changes in the developing tumor. These changes not only result in abnormally proliferating host cells with a heterotrophic and transport-dependent metabolism, but also cause differentiation and serve as mechanisms to balance pathogen defense and adapt to abiotic stress conditions, thereby allowing the coexistence of the crown gall and host plant.
Applied and Environmental Microbiology, 1991
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 di...
Binding of Agrobacterium tumefaciens to carrot protoplasts
Physiological Plant Pathology, 1982
Agrobacterium tumefaciens attached both to intact carrot cells in vitro and to carrot protoplasts. After attachment to the protoplast membrane the bacteria elaborated cellulose fibrils, which anchored them to the surface of the plant cell. These fibrils entrapped other bacteria forming large bacterial clusters on the plant cell surface. The kinetics and bacterial strain specificity of attachment to protoplasts were similar to those observed with intact cells. Thus the plant cell membrane may contain a specific receptor for virulent Agrobacterium cells. It is likely that the transfer of tumor-inducing plasmid DNA from the bacterium to the plant cell would be facilitated by the tight binding of large numbers of bacteria to the plant cell membrane.
Genetic engineering in plants has been aided by the use of Agrobacterium tumefaciens, a Gram negative bacterium that allows the introduction of genetic information into cells. This has revolutionized the field, improving cultivars by expressing traits that would not be possible to obtain by classical methods. As such it is a remarkable system that permits to test in vivo the effect of proteins originated from different species in a particular plant. The wild type Agrobacterium carries two types of genes for transformation: oncogenes and those in charge of opinoids biosynthesis that promote the sickness known as crown gall, which consists in a continuous growth due to the lack of control at the cell cycle. The process of transformation by Agrobacterium requires the presence of several genetic elements: (1) The virulent genes from the chromosome of Agrobacterium (chv), (2) The T-DNA which is limited by the left and right borders (3) The virulent genes from the Ti (vir) plasmid that co...