Variation in Binding and Virulence of Agrobacterium tumefaciens Chromosomal Virulence (chv) Mutant Bacteria on Different Plant Species (original) (raw)
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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.
Journal of bacteriology, 1991
Three Agrobacterium tumefaciens mutants with chromosomal mutations that affect bacterial virulence were isolated by transposon mutagenesis. Two of the mutants were avirulent on all hosts tested. The third mutant, Ivr-211, was a host range mutant which was avirulent on Bryophyllum diagremontiana, Nicotiana tabacum, N. debneyi, N. glauca, and Daucus carota but was virulent on Zinnia elegans and Lycopersicon esculentum (tomato). That the mutant phenotype was due to the transposon insertion was determined by cloning the DNA containing the transposon insertion and using the cloned DNA to replace the wild-type DNA in the parent bacterial strain by marker exchange. The transposon insertions in the three mutants mapped at three widely separated locations on the bacterial chromosome. The effects of the mutations on various steps in tumor formation were examined. All three mutants showed no alteration in binding to carrot cells. However, none of the mutants showed any induction of vir genes b...
Applied and Environmental Microbiology, 2001
tumors. An early step in tumor formation is bacterial attachment to the plant cells. AttR mutants failed to attach to wound sites of both legumes and nonlegumes and were avirulent on both groups of plants. AttR mutants also failed to attach to the root epidermis and root hairs of nonlegumes and had a markedly reduced ability to colonize the roots of these plants. However, AttR mutants were able to attach to the root epidermis and root hairs of alfalfa, garden bean, and pea. The mutant showed little reduction in its ability to colonize these roots. Thus, A. tumefaciens appears to possess two systems for binding to plant cells. One system is AttR dependent and is required for virulence on all of the plants tested and for colonization of the roots of all of the plants tested except legumes. Attachment to root hairs through this system can be blocked by the acetylated capsular polysaccharide. The second system is AttR independent, is not inhibited by the acetylated capsular polysaccharide, and allows the bacteria to bind to the roots of legumes.
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.
The Agrobacterium-Plant Cell Interaction. Taking Biology Lessons from a Bug
PLANT PHYSIOLOGY, 2003
Binns AN, Beaupre CE, Dale EM (1995) Inhibition of VirB-mediated transfer of diverse substrates from Agrobacterium tumefaciens by the InQ plasmid RSF1010. J Bacteriol 177: 4890-4899 Bundock P, den Dulk-Ras A, Beijersbergen A, Hooykaas PJJ (1995) Transkingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. EMBO J 14: 3206-3214 Chen X, Stone M, Schlagnhaufer C, Romaine CP (2000) A fruiting body tissue method for efficient Agrobacterium-mediated transformation of Agaricus bisporus. Appl Environ Microbiol 66: 4510-4513 Chilton MD, Drummond MH, Merio DJ, Sciaky D, Montoya AL, Gordon MP, Nester EW (1977) Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11: 263-271 Chilton MD, Que Q (2003) Targeted integration of T-DNA into the tobacco genome at double-strand breaks: new insights on the mechanism of T-DNA integration. Plant Physiol 133: 956-965 Christie PJ (1997) Agrobacterium tumefaciens T-complex transport apparatus: a paradigm for a new family of multifunctional transporters in eubacteria. J Bacteriol 179:3085-3094, Citovsky V, McLean BG, Greene E, Howard E, Kuldau G, Thorstenson Y, Zupan J, Zambryski PC (1992a) Agrobacterium-plant cell interaction: induction of vir genes and T-DNA transfer. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743 de Groot MJ, Bundock P, Hooykaas PJJ, Beijersbergen AG (1998) Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nat Biotechnol 16:839-842; erratum de Groot MJ, Bundock P, Hooykaas PJJ, Beijersbergen AG (1998) Nat Biotechnol 16: 1074 De Neve M, De Buck S, Jacobs A, Van Montagu M, Depicker A (1997) T-DNA integration patterns in co-transformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J 11: 15-29 Deng W, Chen L, Peng WT, Liang X, Sekiguchi S, Gordon MP, Comai L, Nester EW (1999) VirE1 is a specific molecular chaperone for the exported single-stranded-DNA-binding protein VirE2 in Agrobacterium. Mol Microbiol 31: 1795-1807 Douglas CJ, Staneloni RJ, Rubin RA, Nester EW (1985) Identification and genetic analysis of an Agrobacterium tumefaciens chromsomal virulence region. J Bacteriol 161: 850-860 Escobar MA, Civerolo EL, Politito VS, Pinney KA, Dandekar AM (2003) Characterization of oncogene-silenced transgenic plants: implications for Agrobacterium biology and post-transcriptional gene silencing. Mol Plant Pathol 4: 57-65 Escobar MA, Civerolo EL, Summerfelt KR, Dandekar AM (2001) RNAimediated oncogene silencing confers resistance to crown gall tumorigenesis. Proc Natl Acad Sci USA 98: 13437-13442 Fagard M, Vaucheret H (2000) (Trans)gene silencing in plants: How many mechanisms? Annu Rev Plant Physiol Plant Mol Biol 51: 167-194 Friesner J, Britt AB (2003) Ku80-and DNA ligase IV-deficient plants are sensitive to ionizing radiation and defective in T-DNA integration. Plant J 34: 427-440 Gelvin SB (1998) Agrobacterium VirE2 proteins can form a complex with T strands in the plant cytoplasm. J Bacteriol 180: 4300-4302 Gelvin SB (2000) Agrobacterium and plant genes involved in T-DNA transfer and integration. Annu Rev Plant Physiol Plant Mol Biol 51: 223-256 Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the "gene-jockeying" tool. Microbiol Mol Biol Rev 67: 16-37 Goodner B, Hinkle G, Gattung S, Miller N, Blanchard M, Qurollo B, Goldman BS, Cao Y, Askenazi M, Halling H et al. (2001) Genome sequence of the plant pathogen and biotechnology agent Agrobacterium tumefaciens C58. Science 294: 2323-2328 Gouka RJ, Gerk C, Hooykaas PJ, Bundock P, Musters W, Verrips CT, de Groot MJ (1999) Transformation of Aspergillus awamori by Agrobacterium tumefaciens-mediated homologous recombination. Nat Biotechnol 17: 598-601 Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6: 271-282 Hooykaas PJ, Klapwijk PM, Nuti MP, Schilperoort RA, Rorsch A (1977) Transfer of the Agrobacterium tumefaciens Ti plasmid to avirulent agrobacteria and to explanta. J Gen Microbiol 98: 477-484 Hynes MF, Simon R, Puhler A (1985) The development of plasmid-free strains of Agrobacterium tumefaciens by using incompatibility with a Rhizobium meliloti plasmid to eliminate pAtC58. Plasmid 13: 99-105 Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14: 745-750 Johansen LK, Carrington JC (2001) Silencing on the spot: induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol 126: 930-938 Kunik T,
Factor inducing Agrobacterium tumefaciens vir gene expression is present in monocotyledonous plants
Proceedings of the National Academy of Sciences, 1988
Agrobacterium tumefaciens harboring the tumor-inducing Ti plasmid incites crown gall tumor on dicotyledonous species. Upon infection of these plants, Ti plasmid DNA sequence is stably transferred (T-DNA) by unknown mechanisms to plant cells to be integrated into nuclear DNA. The T-DNA processing and transfer require the expression of vir (virulence) genes on the Ti plasmid, which are known to be induced by certain phenolic compounds released from cells at the wounded inoculation site. The results of the present study demonstrate that wheat and oats contain a substance(s) that induces vir gene expression, yet the inducing substance of wheat differs from the phenolic inducers in that it is hydrophilic and has a molecular weight of several thousand. The novel inducer was not detectable in the exudates of seedlings of these plants but was found in an extract from the transition region between shoot and root of the seedlings and also in extracts from the seeds, bran, and germ. This findi...
Virulence and supervirulence of Agrobacterium tumefaciens in woody fruit plants
Physiological and Molecular Plant Pathology, 1998
The ability of two wild-type (C58 and Ach5) and one transconjugant (A281) Agrobacterium tumefaciens strains to incite tumours on some woody fruit species of the Rosaceae, Vitaceae and Rutaceae was determined in comparison to tobacco and tomato plants. Agrobacterium strain A281 has been reported to be supervirulent on tobacco and tomato. Here, the supervirulent phenotype of A281 was clearly shown on tobacco plants but not on two di erent tomato cultivars. On woody fruit hosts, relative virulence induced by each Agrobacterium strain was dependent on the infecting strain and the host species. On Rutaceae, A281 showed a supervirulent phenotype, while on Rosaceae and on grapevine the responses to all Agrobacterium strains were highly variable and supervirulence was never observed. It is suggested that di erent responses observed on di erent host species are probably due to the specificity of the Agrobacterium strain-host interaction. Implications of these results for the concept of supervirulence associated with strain A281 and its disarmed derivatives are discussed.
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.