Abstract
Regions of the Rhizobium meliloti symbiotic plasmid (20 to 40 kilobase pairs long) containing nodulation (nod) genes were transferred to Agrobacterium tumefaciens or Escherichia coli by conjugation. The A. tumefaciens and E. coli transconjugants elicited root hair curling and the formation of ineffective pseudonodules on inoculated alfalfa plants. A tumefaciens elicited pseudonodules formed at a variable frequency, ranging from 15 to 45%, irrespective of the presence of the Ti plasmid. These pseudonodules developed characteristic nodule meristems, and in some nodules, infection threads were found within the interior of nodules. Infrequently, infection threads penetrated deformed root hairs, but these threads were found only in a minority of nodules. There was no evidence of bacterial release from the infection threads. In addition to being found within threads, agrobacteria were also found in intercellular spaces and within nodule cells that had senesced . In the latter case, the bacteria appeared to invade the nodule cells independently of infection threads and degenerated at the same time as the senescing host cells. No peribacteroid membranes enclosed any agrobacteria , and no bacteroid differentiation was observed. In contrast to the A. tumefaciens-induced pseudonodules , the E. coli-induced pseudonodules were completely devoid of bacteria; infection threads were not found to penetrate root hairs or within nodules. Our results suggest that relatively few Rhizobium genes are involved in the earliest stages of nodulation, and that curling of root hairs and penetration of bacteria via root hair infection threads are not prerequisites for nodule meristem formation in alfalfa.
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Selected References
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- Buikema W. J., Long S. R., Brown S. E., van den Bos R. C., Earl C., Ausubel F. M. Physical and genetic characterization of Rhizobium meliloti symbiotic mutants. J Mol Appl Genet. 1983;2(3):249–260. [PubMed] [Google Scholar]
- Bánfalvi Z., Sakanyan V., Koncz C., Kiss A., Dusha I., Kondorosi A. Location of nodulation and nitrogen fixation genes on a high molecular weight plasmid of R. meliloti. Mol Gen Genet. 1981;184(2):318–325. doi: 10.1007/BF00272925. [DOI] [PubMed] [Google Scholar]
- Corbin D., Barran L., Ditta G. Organization and expression of Rhizobium meliloti nitrogen fixation genes. Proc Natl Acad Sci U S A. 1983 May;80(10):3005–3009. doi: 10.1073/pnas.80.10.3005. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ditta G., Stanfield S., Corbin D., Helinski D. R. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7347–7351. doi: 10.1073/pnas.77.12.7347. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Downie J. A., Ma Q. S., Knight C. D., Hombrecher G., Johnston A. W. Cloning of the symbiotic region of Rhizobium leguminosarum: the nodulation genes are between the nitrogenase genes and a nifA-like gene. EMBO J. 1983;2(6):947–952. doi: 10.1002/j.1460-2075.1983.tb01526.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Eckhardt T. A rapid method for the identification of plasmid desoxyribonucleic acid in bacteria. Plasmid. 1978 Sep;1(4):584–588. doi: 10.1016/0147-619x(78)90016-1. [DOI] [PubMed] [Google Scholar]
- FAHRAEUS G. The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol. 1957 Apr;16(2):374–381. doi: 10.1099/00221287-16-2-374. [DOI] [PubMed] [Google Scholar]
- Forrai T., Vincze E., Bánfalvi Z., Kiss G. B., Randhawa G. S., Kondorosi A. Localization of symbiotic mutations in Rhizobium meliloti. J Bacteriol. 1983 Feb;153(2):635–643. doi: 10.1128/jb.153.2.635-643.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Friedman A. M., Long S. R., Brown S. E., Buikema W. J., Ausubel F. M. Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene. 1982 Jun;18(3):289–296. doi: 10.1016/0378-1119(82)90167-6. [DOI] [PubMed] [Google Scholar]
- Garfinkel D. J., Simpson R. B., Ream L. W., White F. F., Gordon M. P., Nester E. W. Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell. 1981 Nov;27(1 Pt 2):143–153. doi: 10.1016/0092-8674(81)90368-8. [DOI] [PubMed] [Google Scholar]
- Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirsch A. M., Bang M., Ausubel F. M. Ultrastructural analysis of ineffective alfalfa nodules formed by nif::Tn5 mutants of Rhizobium meliloti. J Bacteriol. 1983 Jul;155(1):367–380. doi: 10.1128/jb.155.1.367-380.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hirsch A. M., Long S. R., Bang M., Haskins N., Ausubel F. M. Structural studies of alfalfa roots infected with nodulation mutants of Rhizobium meliloti. J Bacteriol. 1982 Jul;151(1):411–419. doi: 10.1128/jb.151.1.411-419.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
- Hooykaas P. J., Snijdewint F. G., Schilperoort R. A. Identification of the Sym plasmid of Rhizobium leguminosarum strain 1001 and its transfer to and expression in other rhizobia and Agrobacterium tumefaciens. Plasmid. 1982 Jul;8(1):73–82. doi: 10.1016/0147-619x(82)90042-7. [DOI] [PubMed] [Google Scholar]
- JORDAN D. C., GRINYER I., COULTER W. H. ELECTRON MICROSCOPY OF INFECTION THREADS AND BACTERIA IN YOUNG ROOT NODULES OF MEDICAGO SATIVA. J Bacteriol. 1963 Jul;86:125–137. doi: 10.1128/jb.86.1.125-137.1963. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mandel M., Higa A. Calcium-dependent bacteriophage DNA infection. J Mol Biol. 1970 Oct 14;53(1):159–162. doi: 10.1016/0022-2836(70)90051-3. [DOI] [PubMed] [Google Scholar]
- Paau A. S., Bloch C. B., Brill W. J. Developmental fate of Rhizobium meliloti bacteroids in alfalfa nodules. J Bacteriol. 1980 Sep;143(3):1480–1490. doi: 10.1128/jb.143.3.1480-1490.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Patel J. J., Yang A. F. Light and electron microscopic studies of nodule structure of alfalfa. Can J Microbiol. 1981 Jan;27(1):36–43. doi: 10.1139/m81-006. [DOI] [PubMed] [Google Scholar]
- Prakash R. K., Van Brussel A. A., Quint A., Mennes A. M., Schilperoort R. A. The map position of Sym-plasmid regions expressed in the bacterial and endosymbiotic form of Rhizobium leguminosarum. Plasmid. 1982 May;7(3):281–286. doi: 10.1016/0147-619x(82)90009-9. [DOI] [PubMed] [Google Scholar]
- Riedel G. E., Ausubel F. M., Cannon F. C. Physical map of chromosomal nitrogen fixation (nif) genes of Klebsiella pneumoniae. Proc Natl Acad Sci U S A. 1979 Jun;76(6):2866–2870. doi: 10.1073/pnas.76.6.2866. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Robertson J. G., Lyttleton P., Bullivant S., Grayston G. F. Membranes in lupin root nodules. I. The role of Golgi bodies in the biogenesis of infection threads and peribacteroid membranes. J Cell Sci. 1978 Apr;30:129–149. doi: 10.1242/jcs.30.1.129. [DOI] [PubMed] [Google Scholar]
- Rosenberg C., Boistard P., Dénarié J., Casse-Delbart F. Genes controlling early and late functions in symbiosis are located on a megaplasmid in Rhizobium meliloti. Mol Gen Genet. 1981;184(2):326–333. doi: 10.1007/BF00272926. [DOI] [PubMed] [Google Scholar]
- Ruvkun G. B., Ausubel F. M. A general method for site-directed mutagenesis in prokaryotes. Nature. 1981 Jan 1;289(5793):85–88. doi: 10.1038/289085a0. [DOI] [PubMed] [Google Scholar]
- Ruvkun G. B., Ausubel F. M. Interspecies homology of nitrogenase genes. Proc Natl Acad Sci U S A. 1980 Jan;77(1):191–195. doi: 10.1073/pnas.77.1.191. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ruvkun G. B., Sundaresan V., Ausubel F. M. Directed transposon Tn5 mutagenesis and complementation analysis of Rhizobium meliloti symbiotic nitrogen fixation genes. Cell. 1982 Jun;29(2):551–559. doi: 10.1016/0092-8674(82)90171-4. [DOI] [PubMed] [Google Scholar]
- Scott K. F., Hughes J. E., Gresshoff P. M., Beringer J. E., Rolfe B. G., Shine J. Molecular cloning of Rhizobium trifolii genes involved in symbiotic nitrogen fixation. J Mol Appl Genet. 1982;1(4):315–326. [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Truchet G., Rosenberg C., Vasse J., Julliot J. S., Camut S., Denarie J. Transfer of Rhizobium meliloti pSym genes into Agrobacterium tumefaciens: host-specific nodulation by atypical infection. J Bacteriol. 1984 Jan;157(1):134–142. doi: 10.1128/jb.157.1.134-142.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vogelstein B., Gillespie D. Preparative and analytical purification of DNA from agarose. Proc Natl Acad Sci U S A. 1979 Feb;76(2):615–619. doi: 10.1073/pnas.76.2.615. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watson B., Currier T. C., Gordon M. P., Chilton M. D., Nester E. W. Plasmid required for virulence of Agrobacterium tumefaciens. J Bacteriol. 1975 Jul;123(1):255–264. doi: 10.1128/jb.123.1.255-264.1975. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wong C. H., Pankhurst C. E., Kondorosi A., Broughton W. J. Morphology of root nodules and nodule-like structures formed by Rhizobium and Agrobacterium strains containing a Rhizobium meliloti megaplasmid. J Cell Biol. 1983 Sep;97(3):787–794. doi: 10.1083/jcb.97.3.787. [DOI] [PMC free article] [PubMed] [Google Scholar]