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TnphoA-induced symbiotic mutants of Bradyrhizobium japonicum that impair cell and tissue differentiation in Glycine max nodules

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Abstract

Bradyrhizobium japonicum WM1, an ethylmethane-sulfonate-induced derivative of B. japonicum 110spc4 with reduced phosphatase activity but normal symbiotic properties, was randomly mutagenized using TnphoA. From about 1000 purified single colonies, approximately 300, preferentially those with enhanced phosphatase activity, were inoculated onto soybean seedlings to test their symbiotic traits. Sixteen strains were either completely Fix− or possessed markedly reduced acetylene reduction activity (Fixred). Contrary to expectations, hybridization of total DNA of these strains to a transposonspecific DNA probe showed that many contained no transposon. Apparently these strains had gained resistance towards kanamycin spontaneously rather than through the introduction of TnphoA. However, in five mutant strains, two hybridizing BamHI fragments of different sizes were detected, as expected. All strains performed acetylene reduction under ex planta conditions, indicating that mutations had not occurred in nif or fix genes. A more than 50-fold increased specific activity of alkaline phosphatase was observed in strain 132, indicating the synthesis and secretion of a polypeptide fused to 'PhoA. Light and electron-microscopic analyses showed that in nodules induced by strain 132 (Fixred) the infected cells of the central tissue were vacuolated. In some of these cells callose deposition was observed, indicating plant defense reactions. Nodules induced by mutant 184 were infected by bacteroids only in a few cells of the central tissue as isolated clusters, whereas the majority of cells remained uninfected. The concentration of phosphoenolpyruvatecarboxylase protein within the infected tissue was significantly reduced and starch granules accumulated. In both strains TnphoA insertions were identified to be the reasons for the observed phenotypes. These mutant strains should be helpful for studying the influence of the microsymbiont on the differentiation and colonization of infected cells in soybean nodules.

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Abbreviations

dpi:

days post inoculation

EPS:

exopolysac charide (s)

PBM:

peribacteroid membrane

PEP:

phosphoenolpyruvate

PHBA:

polyhydroxybutyric acid

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Author notes

  1. Eva Wegel

    Present address: Max Planck Institut für Züchtungsforschung Abteilung Biochemie, Carl-von-Linné-Weg 10, D-50829, Köln, 30, Germany

Authors and Affiliations

  1. Philipps-Universität Marburg, karl-von-Frisch-Strasse, D-35032, Marburg, Germany

    Peter Müller, Anja Klaucke & Eva Wegel

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  1. Peter Müller
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  2. Anja Klaucke
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Additional information

We thank C. Manoil, Boston, USA, for providing TnphoA. Sirofluor for callose detection was a gift from K. Niehaus, Bielefeld, Germany, the PEP-carboxylase antibody was kindly provided by K. Schuller, Flinders University, Adelaide, Australia. To D. Weiss (Harvard Medical School Boston, Mass., USA) we owe many thanks for corrections of the manuscript and for critical comments. M. Parniske and other collegues in the laboratory have contributed with helpful discussions. This work was supported from a grant of the Deutsche Forschungsgemeinschaft and by a Human Frontiers of Science Project (HFSP) Award to D. Werner.

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Müller, P., Klaucke, A. & Wegel, E. TnphoA-induced symbiotic mutants of Bradyrhizobium japonicum that impair cell and tissue differentiation in Glycine max nodules. Planta 197, 163–175 (1995). https://doi.org/10.1007/BF00239953

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