Summary
A 14 kb DNA fragment from the Sym plasmid of the Rhizobium trifolii strain ANU843, known to carry common nodulation nod and host specific nodulation hsn genes, was extensively mutagenised with transposon Tn5. A correlation between the site of Tn5 insertion and the induced nodulation defect led to the identification of three specific regions (designated I, II, III) which affected nodulation ability. Twenty-three Tn5 insertions into region I (ca. 3.5 kb) affected normal root hair curling ability and abolished infection thread formation. The resulting mutants were unable to nodulate all tested plant species. Tn5 insertions in regions II and III resulted in mutants which showed an exaggerated root hair curling (Hac++) response on clover plants. Ten region II mutants which occurred over a 1.1 kb area showed a greatly reduced nodulation ability on clovers and produced aborted, truncated infection threads. Tn5 insertions into region III (ca. 1.5 kb) altered the outcome of crucial early plant recognition and infection steps by R. trifolii. Seven region III mutants displayed host-range properties which differed from the original parent strain. Region III mutants were able to induce marked root hair distortions, infection threads, and nodules on Pisum sativum including the recalcitrant Afghanistan variety. In addition region III mutants showed a poor nodulation ability on Trifolium repens even though the ability to induce infection threads was retained on this host. The altered host-range properties of region III mutants could only be revealed by mutation and the mutant phenotype was shown to be recessive.
Similar content being viewed by others
References
Banfalvi Z, Sakanyan Y, Koncz C, Kiss A, Dusha I, Kondorosi A (1981) Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid in R. meliloti. Mol Gen Genet 184:318–325
Brewin NJ, Beringer JE, Johnston AWB (1980) Plasmid-mediated transfer of host-range specificity. J Gen Microbiol 120:413–420
Buchanan-Wollaston AV, Beringer JE, Brewin NJ, Hirsch PR, Johnston AWB (1980) Isolation of symbiotically defective mutants in Rhizobium leguminosarum by insertion of the transposon Tn5 into a transmissible plasmid. Mol Gen Genet 178:185–190
Callaham DA, Torrey JG (1981) A structural basis for infection of root hairs of Trifolium repens by Rhizobium. Can J Bot 59:1647–1664
Ditta G, Stanfield S, Corbin D, Helinski DR (1980) Broad host range DNA cloning system for Gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci USA 77:7347–7351
Djordjevic MA, Zurkowski W, Rolfe BG (1982) Plasmids and stability of symbiotic properties of Rhizobium trifolii. J Bacteriol 151:560–568
Djordjevic MA, Zurkowski W, Shine J, Rolfe BG (1983) Sym plasmid transfer to symbiotic mutants of Rhizobium trifolii, Rhizobium leguminosarum and Rhizobium meliloti. J Bacteriol 156:1035–1045
Djordjevic MA, Schofield PR, Ridge RW, Morrison NA, Bassam BJ, Plaszinski J, Watson JM, Rolfe BG (1985) Rhizobium nodulation genes involved in root hair curling (Hac) are functionally conserved. Plant Mol Biol 4:147–160
Downie JA, Hombrecher G, Ma QS, Knight CD, Wells B, Johnston AWB (1983) Cloned nodulation genes of Rhizobium leguminosarum determine host-range specificity. Mol Gen Genet 190:359–365
Downie JA, Knight CD, Johnston AWB, Rossen L (1985) Identification of genes and gene products involved in the nodulation of peas by Rhizobium leguminosarum. Mol Gen Genet 198:255–262
Egelhoff TT, Fischer RF, Jacobs TW, Mulligan JT, Long SR (1985) Nucleotide sequence of Rhizobium meliloti 1021 nodulation genes: nodD is read divergently from nodA,B,C. DNA 4:241–248
Hombrecher G, Brewin NJ, Johnston AWB (1981) Cloning and mutagenesis of nodulation genes from Rhizobium leguminosarum TOM, a strain with extended host range. Mol Gen Genet 182:767–770
Hooykaas PJJ, Hofker M, den Dulk-Ras H, Schilperoort RA (1984) A comparison of virulence determinants in an octipine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants. Plasmid 11:195–205
Kondorosi E, Banfalvi Z, Kondorosi A (1984) Physical and genetic analysis of a symbiotic region of Rhizobium meliloti: identification of nodulation genes. Mol Gen Genet 193:445–452
Rolfe BG, Gresshoff PM, Shine J (1980) Rapid screening for symbiotic mutants of Rhizobium and white clover. Plant Sci Lett 19:277–284
Rolfe BG, Djordjevic M, Scott KF, Hughes JE, Badenoch-Jones J, Gresshoff PM, Cen Y, Dudman WF, Zurkowski W, Shine J (1981) Analysis of nodule-forming ability of fast-growing Rhizobium strains. In: Gibson AH, Newton WE (eds) Current perspecitives in nitrogen fixation. Australian Academy of Sciences, Canberra, pp 142–145
Rolfe BG, Scott KF, Schofield PR, Watson JM, Plazinski J, Djordjevic MA (1985) Genetic and molecular analysis of host range and nodulation genes in Rhizobium trifolii and the Parasponia Rhizobium strain ANU289. In: Szalay AA, Legocki RP (eds) Advances in the molecular genetics of the bacteria-plant interaction. Media Services Cornell University, Ithica, NY, pp 43–48
Rosenberg C, Boistard P, Denarie J, Casse-Delbart FL (1981) Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol Gen Genet 184:326–333
Rossen L, Johnston AWB, Downie JA (1984) DNA sequence of the Rhizobium leguminosarum nodulation genes nodA.B and C required for root hair curling. Nucleic Acids Res 12:9497–9508
Ruvkun GB, Ausubel FM (1981) A general method for site-directed mutagenesis in procaryotes. Nature 289:85–88
Schofield PR, Djordjevic MA, Rolfe BG, Shine J, Watson JM (1983) A molecular linkage map of nitrogenase and nodulation genes in Rhizobium trifolii. Mol Gen Genet 192:459–465
Schofield PR, Ridge RW, Rolfe BG, Shine J, Watson JM (1984) Host-specific nodulation is encoded on a 14 kb fragment in Rhizobium trifolii. Plant Mol Biol 3:3–11
Simon R, Priefer U, Puhler A (1982) Vector plasmids for in vivo and in vitro manipulations of Gram-negative bacteria. In: Puhler A (ed) Molecular genetics of the bacteria-plant interaction. Springer, Berlin Heidelberg New York, pp 98–106
Staskawicz BJ, Dahlbeck D, Keen NT (1984) Cloned avirulence gene of Pseudomonas syringae pv. glycinea determines race-specific incompatibility on Glycine max (L) Merr. Proc Natl Acad Sci USA 81:6024–6038
Torok I, Kondorosi E, Stepkowski T, Posfai J, Kondorosi A (1984) Nucleotide sequence of Rhizobium meliloti nodulation genes. Nucleic Acids Res 12:9509–9524
Vasse JM, Truchet GL (1984) The Rhizobium-legume symbiosis: observation of root infection by bright-field microscopy after staining with methylene blue. Planta 161:487–489
Vincent JM (1977) Rhizobium: general microbiology. In: Hardy RWF, Silver WS (eds) A treatise on dinitrogen fixation, vol 3. John Wiley and Sons, New York, pp 277–364
Vincent JM (1980) Factors controlling the legume-Rhizobium symbiosis. In: Newton WE, Orme-Johnston WH (eds) Nitrogen fixation. University Park Press, Baltimore, pp 103–129
Author information
Authors and Affiliations
Additional information
Communicated by M.M. Green
Rights and permissions
About this article
Cite this article
Djordjevic, M.A., Schofield, P.R. & Rolfe, B.G. Tn5 mutagenesis of Rhizobium trifolii host-specific nodulation genes result in mutants with altered host-range ability. Molec. Gen. Genet. 200, 463–471 (1985). https://doi.org/10.1007/BF00425732
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00425732