Abstract
A technique was optimized for thein situ detection of nodulation (nod) gene activity inRhizobium leguminosarum bv.viciae symbiosis with compatible plant hostsVicia tetrasperma (L.)Schreb. andPisum sativum L. The transcription ofnodABC-lacZ fusion was visualized as β-galactosidase (β-Gal) activity after reaction with the chromogenic substrate X-Gal and subsequent light microscopy, while the back-ground of the indigenous β-Gal activity of rhizobia and the host plant was eliminated by glutaraldehyde treatment.V. tetrasperma was suggested as a suitable model plant for pea cross-inoculation group due to its advantages over the common model ofV. hirsuta (L.) S.F.Gray: compactness of the plant, extremely small seeds, fast development and stable nodulation under laboratory conditions. In the roots of both plants, a certain extent ofnod gene activity was detectable in all rhizobia colonizing the rhizoplane. In pea 1 d after inoculation (d.a.i.), the maximum was localized in the region of emerging root hairs (RH) later (3 and 6 d.a.i.) shifting upwards from the root tip. Nodulation genes sustained full expression even in the infection threads inside the RH and the root cortex, independently of their association with nodule primordia. Comparison of two pea symbiotic mutant lines, Risnod25 and Risnod27, with the wild type did not reveal any differences in the RH formation, RH curling response and rhizoplane colonization. Both mutants appeared to be blocked at the infection thread initiation stage and in nodule initiation, consistent with the phenotype caused by other mutant alleles in the peasym8 locus. Judging from thenod gene expression level and pattern in the rhizoplane, flavonoid response upon inoculation is preserved in both pea mutants, being independent of infection thread and nodule initiation.
Similar content being viewed by others
Abbreviations
- d.a.i.:
-
day(s) after inoculation
- GA:
-
glutaraldehyde
- β-Gal:
-
β-galactosidase
- Ini:
-
increased nodulation gene-inducing activity (response)
- IT:
-
infection thread(s)
- Iti:
-
infection thread initiation stage
- Noi:
-
nodule initiation
- RH:
-
root hair(s)
References
Albrecht C., Geurts R., Lapeyrie F., Bisseling T.: Endomycorrhizae and rhizobial Nod factors both require SYM8 to induce the expression of the early nodulin genes PsENOD5 and PsENOD12A.Plant J. 15, 605–614 (1998).
Arséne F., Katupitiya S., Kennedy I.R., Elmerich C.: Use oflacZ fusions to study the expression ofnif genes ofAzospirillum brasilense in association with plants.Mol.Plant-Microbe Interact. 7, 748–757 (1994).
Boivin C., Camut S., Malpica C.A., Truchet G., Rosenberg C.:Rhizobium meliloti genes encoding catabolism of trigonelline are induced under symbiotic conditions.Plant Cell 2, 1157–1170 (1990).
Borisov A.Y., Jacobi L.M., Lebski V.K., Morzhina E.V., Tsyganov V.E., Voroshilova V.A., Tikhonovich T.A.: Genetic system controlling development of nitrogen-fixing nodules and arbuscular mycorrhiza.Pisum Genet. 31, 40–43 (1999).
Burn J., Rossen L., Johnston A.W.B.: Four classes of mutations in thenodD gene ofRhizobium leguminosarum biovarviciae that affect its ability to autoregulate and/or activate othernod genes in the presence of flavonoid inducers.Genes & Dev. 1, 456–464 (1987).
Catoira R., Galera C., De Billy F., Penmetsa R.V., Journet E.-P., Maillet F., Rosenberg C., Cook D., Gough C., Dénarié J.: Four genes ofMedicago truncatula controlling components of a Nod factor transduction pathway.Plant Cell 12, 1647–1665 (2000).
Downie J.A., Walker S.A.: Plant responses to nodulation factors.Curr.Opin.Plant Biol. 2, 483–489 (1999).
Engvild K.C.: Nodulation and nitrogen fixation mutants of pea,Pisum sativum.Theor.Appl.Genet. 74, 711–713 (1987).
Knight C.D., Rossen L., Robertson J.G., Wells B., Downie J.A.: Nodulation inhibition byRhizobium leguminosarum multicopynodABC genes and analysis of early stages of plant infection.J.Bacteriol. 166, 552–558 (1986).
Leyva A., Palacios J.N., Ruiz-Argueso T.: Conserved plasmid hydrogen-uptake (hup)-specific sequences within Hup+ Rhizobium leguminosarum strains.Appl.Environ.Microbiol. 53, 2539–2543 (1987).
Lie T.A.: The effect of low pH on different phases of nodule formation in pea plants.Plant & Soil 31, 391–406 (1969a).
Lie T.A.: Non-photosynthetic effects of red and far-red light on root-nodule formation by leguminous plants.Plant & Soil 30, 391–404 (1969b).
Miller J.H.:Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA) 1972.
Novák K.: Allelic relationships of pea nodulation mutants.J.Heredity 94, 191–193 (2003).
Novák K., Škrdleta V., Němcová M., Lisá L.: Optimization of rhizobialnod gene-inducing activity assay in pea root exudate.Folia Microbiol. 39, 208–214 (1994).
Novák K., Chovanec P., Škrdleta V., Kropáčová M., Lisá L., Němcová M.: Effect of exogenous flavonoids on nodulation of pea (Pisum sativum L.).J.Exp.Bot. 53, 1735–1745 (2002).
Novák K., Lisá L., Škrdleta V.: Rhizobialnod gene-inducing activity in pea nodulation mutants: dissociation of nodulation and flavonoid response.Physiol.Plant. 120, 546–555 (2004).
Oke V., Long S.R.: Bacterial genes induced within the nodule during theRhizobium-legume symbiosis.Mol.Microbiol. 32, 837–849 (1999).
Oldroyd G.E.D., Engstrom E.M., Long S.R.: Ethylene inhibits the Nod factor signal transduction pathway ofMedicago truncatula.Plant Cell 13, 1835–849 (2001).
Penmetsa R.V., Cook D.R.: A legume ethylene-insensitive mutant hyperinfected by its rhizobial symbiont.Science 275, 527–530 (1997).
Quandt H.J., Puhler A., Broer I.: Transgenic root nodules ofVicia hirsuta — a fast and efficient system for the study of gene expression in indeterminate-type nodules.Mol.Plant-Microbe Interact. 6, 699–706 (1993).
Recourt K., Schripsema J., Kijne J.W., Van Brussel A.A.N., Lugtenberg B.J.J.: Inoculation ofVicia sativa subsp.nigra roots withRhizobium leguminosarum biovarviciae results in release ofnod gene activating flavanones and chalcones.Plant Mol.Biol. 16, 841–852 (1991).
Redmont J.W., Batley M., Djordjevic M.A., Innes R.W., Kuempel P.L., Rolfe B.G.: Flavones induce expression of nodulation genes inRhizobium.Nature 323, 632–635 (1986).
Sagan M., Huguet T., Duc G.: Phenotypic characterization and classification of nodulation mutants of pea (Pisum sativum L.).Plant Sci. 100, 59–70 (1994).
Sambrook J., Fritsch E.F., Maniatis T.:Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (USA) 1989.
Schlaman H.R.M., Horvath B., Vijgenboom E., Okker R.J.H., Lugtenberg B.J.J.: Suppression of nodulation gene expression in bacteroids ofRhizobium leguminosarum biovarviciae.J.Bacteriol. 173, 4277–4287 (1991).
Schneider A., Walker S.A., Sagan M., Duc G., Ellis T.H.N., Downie J.A.: Mapping of the nodulation locisym9 andsym10 of pea (Pisum sativum L.).Theor.Appl.Genet. 104, 1312–1316 (2002).
Sharma S.B., Signer E.R.: Temporal and spatial regulation of the symbiotic genes ofRhizobium meliloti in planta revealed by transposon Tn5-gusA.Genes & Dev. 4, 344–356 (1990).
Shen S.C., Wang S.P., Yu G.Q., Zhu J.B.: Expression of the nodulation and nitrogen fixation genes inRhizobium meliloti during development.Genome 31, 354–360 (1989).
Spaink H.P.: Root nodulation and infection factors produced by rhizobial bacteria.Ann.Rev.Microbiol. 54, 257–288 (2000).
Spaink H.P., Wijffelman C.A., Pees E., Okker R.J.H., Lugtenberg B.J.J.:Rhizobium nodulation genenodD as a determinant of host specificity.Nature 328, 337–340 (1987a).
Spaink H.P., Okker R.J.H., Wijffelman C.A., Pees E., Lugtenberg B.J.J.: Promoters in the nodulation region of theRhizobium leguminosarum Sym plasmid pRL1JI.Plant Mol.Biol. 9, 27–39 (1987b).
Spaink H.P., Okker R.J.H., Wijffelman C.A., Tak T., Goosen-de Roo L., Pees E., Van Brussel A.A.N., Lugtenberg B.J.J.: Symbiotic properties of rhizobia containing a flavonoid-independent hybridnodD product.J.Bacteriol. 171, 4045–4053 (1989).
Stracke S., Kistner C., Yoshida S., Mulder L., Sato S., Kaneko T., Tabata S., Sandal N., Stougaard J., Szczyglowski K., Parniske M.: A plant receptor-like kinase required for both bacterial and fungal symbiosis.Nature 417, 959–962 (2002).
Tsyganov V.E., Voroshilova V.A., Priefer U.B., Borisov A.Y., Tikhonovich I.A.: Genetic dissection of the infection process and nodule tissue development in theRhizobium-pea (Pisum sativum L.) symbiosis.Ann.Bot. 89, 357–366 (2002).
Van Brussel A.A.N., Tak T., Wetselaar A., Pees E., Wijffelman C.A.: SmallLeguminosae as test plants for nodulation ofRhizobium leguminosarum and other rhizobia and agrobacteria harboring aleguminosarum Sym-plasmid.Plant Sci.Lett. 27, 317–325 (1982).
Vincent J.M.:A Manual for the Practical Study of the Root Nodule Bacteria. Blackwell, Oxford (UK) 1970.
Voroshilova V.A., Boesten B., Tsyganov V.E., Borisov A.Y., Tikhonovich I.A., Priefer U.B.: Effect of mutations inPisum sativum L. genes blocking different stages of nodule development on the expression of late symbiotic genes inRhizobium leguminosarum bv.viciae.Mol.Plant-Microbe Interact. 14, 471–476 (2001).
Wilson K.J.: Molecular techniques for the study of rhizobial ecology in the field.Soil.Biol.Biochem. 27, 501–514 (1995).
Xi C., Dirix G., Hofkens J., De Schryver F.C., Vanderleyden J., Michiels J.: Use of dual marker transposons to identify new symbiosis genes inRhizobium.Microbial Ecol. 41, 325–332 (2001).
Yang W.C., Cremers H.C.J.C., Hogendijk P., Katinakis P., Wijffelman C.A., Franssen H., Van Kammen A., Bisseling T.:In situ localization of chalcone synthase messenger RNA in pea root nodule development.Plant J. 2, 143–151 (1992).
Zaat S.A.J., Wijffelman C.A., Mulders I.H.M., Van Brussel A.A.N., Lugtenberg B.J.J.: Root exudates of various host plants ofRhizobium leguminosarum contain different sets of inducers ofRhizobium nodulation genes.Plant Physiol. 86, 1298–1303 (1988).
Author information
Authors and Affiliations
Additional information
The work was supported by grant no. 521/03/0192 of theGrant Agency of the Czech Republic and by theInstitutional Research Concept AV 0Z 5020 0510.
Rights and permissions
About this article
Cite this article
Chovanec, P., Novák, K. Visualization of nodulation gene activity on the early stages ofRhizobium leguminosarum bv.viciae symbiosis. Folia Microbiol 50, 323–331 (2005). https://doi.org/10.1007/BF02931413
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF02931413