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Excision of Nodules Induced by Rhizobium meliloti Exopolysaccharide Mutants Releases Autoregulation in Alfalfa
JPlantPhysiol. Vol. 138.pp. 765-767(1991}
Short Comnlunication
Excision of Nodules Induced by Rhizobium meliloti Exopolysaccharide Mutants Releases Autoregulation in Alfalfa GUSTAVO CAETANO-ANOLLES
and PETER M.
GRESSHOFF
Plant Molecular Genetics (OHLD), Institute of Agriculture and Center for Legume Research, The University of Tennessee, Knoxville, Tennessee 37901-1071, USA Received February 26,1991 . Accepted May 18, 1991
Summary Nodulation in alfalfa (Medicago sativa L.) is controlled by a systemic feedback regulatory mechanism that suppresses nodule initiation in younger portions of the root system. Excision of primary root nodules induced by wild-type Rhizobium meliloti stimulates the formation of new nodules on lateral roots. In similar experiments we found that excision of bacteria-free nodules from primary roots induced by mutants of R. meliloti deficient in exopolysaccharide synthesis allowed nodules to reappear in lateral roots especially around the root tip at the time of nodule removal. Our results suggest that organized nodular structures trigger autoregulatory responses in legumes, even in the absence of bacterial infection.
Key words: Autoregulation, feedback control, Medicago sativa, nodulation, Rhizobium meliloti. Abbreviations: EH
=
smallest emergent root hair; EPS
Introduction Organized nodular structures can be formed without infection in alfalfa. For example, ineffective bacteria-free nodules are induced by R. meliloti mutants deficient in exopolysaccharide synthesis (exo mutants; Finan et al., 1985; Leigh et al., 1987) and Agrobacterium tume/aciens transconjugants carrying R. meliloti nodulation genes (Wong et al., 1983; Truchet et al., 1984; Hirsch et al., 1984). These structures resemble normal indeterminate nodules with a central tissue surrounded by nodule parenchyma, an endoderm is and a nodule cortex, and containing peripheral vascular bundles (Van de Wiel et al., 1990). Similar structures are induced in the absence of Rhizobium, either by treatment with auxintransport inhibitors (Hirsch et al., 1989) or extracellular nodulation signals (Lerouge et al., 1990), when Rhizobium is separated from the roots by filter membranes (Kapp et al., 1990), or spontaneously (Truchet et al., 1989; CaetanoAnolles et al., 1991 a). The formation of nodules in alfalfa is controlled by a systemic feedback regulatory mechanism that suppresses nodulation in younger portions of the root system (CaetanoAnolIes and Bauer, 1988). Nodule excision experiments dem© 1991 by Gustav Fischer Verlag, Stuttgart
=
exopolysaccharide; RT
=
root tip.
onstrated that feedback suppression was exerted at the level of nodule initiation rather than during infection development (Caetano-Anolles and Gresshoff, 1991). In a series of split-root assays, nodules induced by R. meliloti exo mutants were able to elicit and be the target of the systemic response (Caetano-Anolles and Bauer, 1990). Similarly, we demonstrated that spontaneous nodules induced in the absence of Rhizobium elicit feedback regulation (Caetano-Anolles et al., 1991 a). Here we show that excision of nodules formed by exo mutants releases the systemic response that controls nodulation.
Materials and Methods Rhizobium meliloti 1021 is a streptomycin-resistant derivative of strain RCR2011 (also known as SU 47). The exoB mutant derivative Rm7094 is unable to synthesize exopolysaccharides EPS I and EPS II, and the exoF mutant Rm7055 and the exoA mutant Rm7061 are defective in EPS I (Leigh et al., 1985). All strains were originally obtained from J. A. Leigh, University of Washington, Seattle, Washington, USA. Bacteria were maintained and grown as described (Caetano-Anolles et al., 1990).
766
GUSTAVO CAETANO-ANOLLES and PETER M. GRESSHOFF
Alfalfa (Medicago sativa L. cv. Vernal) seeds were provided by R. Van Keuren, Agronomy Department, Ohio State University, Wooster, Ohio, USA. Seeds were surface sterilized with ethanol and mercuric chloride and germinated in water-agar plates (CaetanoAnolles et al., 1990). Two-day-old seedlings were transferred in groups of 5 to ethylene-oxide-sterilized plastic growth pouches (Northrup King Seed Co., Minneapolis, MN, USA) containing 10 mL of nitrogen-free Jensen solution Oensen, 1942). Primary roots were inoculated 3 d later with 100 ILL of a bacterial suspension containing 106 cells diluted from a late exponential growth-phase culture. At the time of inoculation, the positions of the root tip (RT) and of the smallest emergent root hairs (EH) were marked on the plastic surface of the pouch with the aid of a dissecting microscope at 12 x magnification. The plants were cultured in a growth chamber at 70-80% relative humidity, 25°C day/22°C night temperatures, a 16-h photoperiod, and 500 Ilmol· s - I • m - 2 (PPF). Nodules were counted at regular intervals following inoculation. In some cases, nodules were carefully excised with a sterile scalpel and retrieved from the pouch. The position of the root tip of each lateral root was marked at the time of excision and the number and location of individual nodules determined at the end of the experiment both on primary and lateral roots with a computer-linked graphics tablet equipped with a dual-action digitizing pen (IS/One graphics pad; Kurta Corp., Phoenix, Arizona).
Results and Discussion
Nodule formation in alfalfa is developmentally restricted to the region of emerging root hairs (Bhuvaneswari et al., 1981; Caetano-AnolIes et al., 1990). Nodulation occurs almost exclusively on the primary root when plants are inoculated before lateral root emergence (Caetano-AnolIes and Gresshoff, 1991). In particular, inoculation of the plants with R. meliloti RCR2011 at day 5 produces the earliest appearance of nodules and emergence at maximal rate. In this study, the antibiotic-resistant derivative strain 1021 behaved in the same way, but several exo mutant derivatives of 1021 formed abundant nodules on lateral roots. Fig. 1 A shows the kinetics of nodule development in primary and lateral roots when plants were inoculated with a R. meliloti exoB mutant. Nodules first appeared on the primary root 4 d after inoculation and emerged at a constant rate during the following 4 to 5 d. Very few if any additional nodules appeared on the primary root once the nodulation plateau was reached or within an additional month of cultivation. Nodules on lateral roots emerged 12 d after inoculation with a constant rate of up to 0.8 ± 0.05 (SD) nodules' d -I. When fully developed nodules induced by wild-type or exo mutants were excised and removed from the plastic growth pouch, new nodules were formed on lateral roots. In plants inoculated with exo mutants, a significant increase in both the number of lateral root nodules and the rate of nodule emergence was observed. After nodule excision, the rate of nodulation induced by an exoB mutant on lateral roots doubled (Fig. 1) but never reached the levels obtained when nodules induced by parent R. meliloti 1021 were excised (3.8 ± 0.4 nodules' d -I). Delays in excision had no effect on the rate of nodule formation (Fig. 1). However, the number of nodules formed on lateral roots at the end of the experiment doubled when nodules were excised 12 d post-inoculation but only increased 30 - 40 % when excision was delayed by a
20
A
Lateral roots
I
10
Primary root
30
B
IZ
c:( -I
a..
20
C/)
w
-I
::)
Cl 10
0 Z
20
C
10
DAYS AFTER INOCULATION
Fig. 1: Effect of nodule excision on subsequent nodule emergence. Sets of 47 to 59 plants in groups of five were inoculated with 2 x 106 bacteria/plant of R. meliloti Rm7094 (exoB94:Tn5) 5 d after seed imbibition, and the appearance of nodules on the primary root (0) and on lateral roots (e) was determined at regular intervals. Nodules were either left undisturbed (A) or were excised 12 d (B) or 19 d after inoculation (C). Bars indicate maximum 95 % confidence limits. The results shown are representative of 2 independent experiments. Primary roots reached the bottom of the growth pouch 10 to 12 d after germination.
week. Since no inherent limitation on lateral root nodulation was observed with time (Caetano-Anolles and Gresshoff, 1991), the difference results from the constraints of our experimental design. While very few if any new nodules emerged on the primary root (Fig. 1 Band q, new nodules appeared on the lateral roots particularly in the vicinity of the lateral RT mark made at the time of excision (data not shown). This region of the lateral roots was the one susceptible to infection at the time of nodule removal. Nutman (1952) found that surgical excision of functional nodules or root tips stimulated the formation of new nodules in red clover. This result was interpreted as the ability of established meristematic foci to inhibit new nodule formation and showed that the extent of nodulation was regulated by the host plant. We found that excision of first nodules releases the autoregulatory suppression of nodulation in alfalfa and soybean. Removal of fully developed nodules from alfalfa primary roots caused nodules to emerge almost ex-
Rhizobium meliloti exo mutants control nodulation in alfalfa
elusively around the location of the lateral root tips at the time of nodule excision (Caetano-Anolles and Gresshoff, 1991). In soybean, nodule excision resulted in new nodules emerging on primary and lateral roots in those same regions from where nodules were initially removed and from infections that were already initiated at the time (Caetano-Anolles et al., 1991 b). The different location in nodule emergence indicates the existence of at least two alternative mechanisms that control nodulation in legumes, one favoring multiplicity of infections and the selective arrest of nodule development, the other controlling nodule initiation. Our present studies demonstrate that the excision of bacteria-free nodules induced by exo mutants stimulates the development of additional nodules. Split-root experiments have already shown that these empty nodules elicit the systemic autoregulatory response that controls nodulation (Caetano-Anolles et al., 1990). However, nodule excision experiments in soybean revealed nodulation control mechanisms other than systemic regulatory responses (Caetano-AnolIes et al. 1991 b). It was then important to determine if nodule excision released nodulation control even in the absence of bacterial infection. The observation that nodule removal stimulates nodulation suggests further that organized nodular structures devoid of inter- or intracellular bacteria are active in controlling nodulation. Since approachgrafting of a soybean mutant blocked in nodule development to its parent showed that cortical cell division without infection triggered the systemic regulatory response (CaetanoAnolIes and Gresshoff, 1990) and spontaneous alfalfa nodules formed in the absence of Rhizobium induced autoregulation (Caetano-AnolIes et al., 1991 a), our results suggest that the plant plays a key role in regulation of nodule number. In particular, the existence of an homeostatic control of nodule number as the one reported here can be of relevance in agronomical situations where there is nodule loss by desiccation or defoliation at the time of alfalfa harvesting. Acknowledgements This work was supported by an endowment to the Racheff Chair of Excellence of The University of Tennessee, and the Tennessee Soybean Promotion Board.
767
References BHUVANESWARI, T. v., A. A. BHAGWAT, and W. D. BAUER: Plant Physiol. 68,1144-1149 (1981). CAETANO-ANOLLES, G. and W. D. BAUER: Plant a 175, 546-557 (1988). CAETANO-ANOLLES, G. and P. M. GRESSHOFF: Plant Sci. 71, 69-81 (1990). - - Plant Physiol. 95, 366-373 (1991). CAETANO-ANOLLES, G., A. LAGARES, and W. D. BAUER: Plant Physiol. 92, 368-374 (1990). CAETANO-ANOLLES, G., J. A. JOSHI, and P. M. GRESSHOFF: Planta 183, 77 -82 (1991 a). CAETANO-ANOLLES, G., E. T. PAPAROZZI, and P. M. GRESSHOFF: J. Plant Physiol. 137, 389-396 (1991 b). FINAN, T. M., A. M. HIRSCH, J. A. LEIGH, E. JOHANSEN, G. A. KULDAU, S. DEEGAN, G. C. WALKER, and E. R. SIGNER: Cell 40, 869-877 (1985). HIRSCH, A. M., T. V. BHUVANESWARI, T. G. TORREY, and T. BISSELING: Proc. Natl. Acad. Sci. USA 86,1244-1248 (1989). HIRSCH, A. M., K. J. WILSON, J. D. G. JONES, M. BANG, V. V. WALKER, and F. M. AUSUBEL: J. Bacteriol. 158, 1133-1143 (1984). JENSEN, H. L.: Proc. Linn. Soc. N. S. W. 66, 98-108 (1942). KApP, D., K. NIEHAUS, J. QUANDT, P. MULLER, and A. PUHLER: Plant Cell 2, 139-151 (1990). LEIGH, J. A., E. R. SIGNER, and G. C. WALKER: Proc. Natl. Acad. Sci. USA 82,6231-6235 (1985). LEIGH, J. A., J. W. REED, J. F. HANKS, A. M. HIRSCH, and G. C. WALKER: Cell 51, 579-587 (1987). LEROUGE, P., P. ROCHE, J. PROME, C. FAUCHER, J. VASSE, F. MAILLET, S. CAMUT, F. DE BILLY, D. G. BARKER, J. DENARIE, and G. TRUCHET: In: GRESSHOFF, P. M., E. ROTH, G. STACEY, and W. E. NEWTON (eds.), Nitrogen fixation: achievements and objectives, pages 177 -186. Chapman and Hall, New York (1990). NUTMAN, P. S.: Ann. Bot. N. S. 16, 81-102 (1952). TRUCHET, G., C. ROSENBERG, J. VASSE, J. S. JULLIOT, S. CAMUT, and J. DENARIE: J. Bacteriol. 157, 134-142 (1984). TRUCHET, G., D. G. BARKER, S. CAMUT, F. DE BILLY, J. VASSE, and T. HUGUET: Mol. Gen. Genet. 219, 65-68 (1989). VAN DE WIEL, c., J. H. NORRIS, B. BOCHENEK, R. DICKSTEIN, T. BISSELING,and A. M. HIRSCH: Plant Cell 2, 1009-1017 (1990). WONG, C. H., C. E. PANKHURST, A. KONDOROSI, and W. J. BROUGHTON: J. Cell. BioI. 97, 787 -794 (1983).
Short Comnlunication
Excision of Nodules Induced by Rhizobium meliloti Exopolysaccharide Mutants Releases Autoregulation in Alfalfa GUSTAVO CAETANO-ANOLLES
and PETER M.
GRESSHOFF
Plant Molecular Genetics (OHLD), Institute of Agriculture and Center for Legume Research, The University of Tennessee, Knoxville, Tennessee 37901-1071, USA Received February 26,1991 . Accepted May 18, 1991
Summary Nodulation in alfalfa (Medicago sativa L.) is controlled by a systemic feedback regulatory mechanism that suppresses nodule initiation in younger portions of the root system. Excision of primary root nodules induced by wild-type Rhizobium meliloti stimulates the formation of new nodules on lateral roots. In similar experiments we found that excision of bacteria-free nodules from primary roots induced by mutants of R. meliloti deficient in exopolysaccharide synthesis allowed nodules to reappear in lateral roots especially around the root tip at the time of nodule removal. Our results suggest that organized nodular structures trigger autoregulatory responses in legumes, even in the absence of bacterial infection.
Key words: Autoregulation, feedback control, Medicago sativa, nodulation, Rhizobium meliloti. Abbreviations: EH
=
smallest emergent root hair; EPS
Introduction Organized nodular structures can be formed without infection in alfalfa. For example, ineffective bacteria-free nodules are induced by R. meliloti mutants deficient in exopolysaccharide synthesis (exo mutants; Finan et al., 1985; Leigh et al., 1987) and Agrobacterium tume/aciens transconjugants carrying R. meliloti nodulation genes (Wong et al., 1983; Truchet et al., 1984; Hirsch et al., 1984). These structures resemble normal indeterminate nodules with a central tissue surrounded by nodule parenchyma, an endoderm is and a nodule cortex, and containing peripheral vascular bundles (Van de Wiel et al., 1990). Similar structures are induced in the absence of Rhizobium, either by treatment with auxintransport inhibitors (Hirsch et al., 1989) or extracellular nodulation signals (Lerouge et al., 1990), when Rhizobium is separated from the roots by filter membranes (Kapp et al., 1990), or spontaneously (Truchet et al., 1989; CaetanoAnolles et al., 1991 a). The formation of nodules in alfalfa is controlled by a systemic feedback regulatory mechanism that suppresses nodulation in younger portions of the root system (CaetanoAnolIes and Bauer, 1988). Nodule excision experiments dem© 1991 by Gustav Fischer Verlag, Stuttgart
=
exopolysaccharide; RT
=
root tip.
onstrated that feedback suppression was exerted at the level of nodule initiation rather than during infection development (Caetano-Anolles and Gresshoff, 1991). In a series of split-root assays, nodules induced by R. meliloti exo mutants were able to elicit and be the target of the systemic response (Caetano-Anolles and Bauer, 1990). Similarly, we demonstrated that spontaneous nodules induced in the absence of Rhizobium elicit feedback regulation (Caetano-Anolles et al., 1991 a). Here we show that excision of nodules formed by exo mutants releases the systemic response that controls nodulation.
Materials and Methods Rhizobium meliloti 1021 is a streptomycin-resistant derivative of strain RCR2011 (also known as SU 47). The exoB mutant derivative Rm7094 is unable to synthesize exopolysaccharides EPS I and EPS II, and the exoF mutant Rm7055 and the exoA mutant Rm7061 are defective in EPS I (Leigh et al., 1985). All strains were originally obtained from J. A. Leigh, University of Washington, Seattle, Washington, USA. Bacteria were maintained and grown as described (Caetano-Anolles et al., 1990).
766
GUSTAVO CAETANO-ANOLLES and PETER M. GRESSHOFF
Alfalfa (Medicago sativa L. cv. Vernal) seeds were provided by R. Van Keuren, Agronomy Department, Ohio State University, Wooster, Ohio, USA. Seeds were surface sterilized with ethanol and mercuric chloride and germinated in water-agar plates (CaetanoAnolles et al., 1990). Two-day-old seedlings were transferred in groups of 5 to ethylene-oxide-sterilized plastic growth pouches (Northrup King Seed Co., Minneapolis, MN, USA) containing 10 mL of nitrogen-free Jensen solution Oensen, 1942). Primary roots were inoculated 3 d later with 100 ILL of a bacterial suspension containing 106 cells diluted from a late exponential growth-phase culture. At the time of inoculation, the positions of the root tip (RT) and of the smallest emergent root hairs (EH) were marked on the plastic surface of the pouch with the aid of a dissecting microscope at 12 x magnification. The plants were cultured in a growth chamber at 70-80% relative humidity, 25°C day/22°C night temperatures, a 16-h photoperiod, and 500 Ilmol· s - I • m - 2 (PPF). Nodules were counted at regular intervals following inoculation. In some cases, nodules were carefully excised with a sterile scalpel and retrieved from the pouch. The position of the root tip of each lateral root was marked at the time of excision and the number and location of individual nodules determined at the end of the experiment both on primary and lateral roots with a computer-linked graphics tablet equipped with a dual-action digitizing pen (IS/One graphics pad; Kurta Corp., Phoenix, Arizona).
Results and Discussion
Nodule formation in alfalfa is developmentally restricted to the region of emerging root hairs (Bhuvaneswari et al., 1981; Caetano-AnolIes et al., 1990). Nodulation occurs almost exclusively on the primary root when plants are inoculated before lateral root emergence (Caetano-AnolIes and Gresshoff, 1991). In particular, inoculation of the plants with R. meliloti RCR2011 at day 5 produces the earliest appearance of nodules and emergence at maximal rate. In this study, the antibiotic-resistant derivative strain 1021 behaved in the same way, but several exo mutant derivatives of 1021 formed abundant nodules on lateral roots. Fig. 1 A shows the kinetics of nodule development in primary and lateral roots when plants were inoculated with a R. meliloti exoB mutant. Nodules first appeared on the primary root 4 d after inoculation and emerged at a constant rate during the following 4 to 5 d. Very few if any additional nodules appeared on the primary root once the nodulation plateau was reached or within an additional month of cultivation. Nodules on lateral roots emerged 12 d after inoculation with a constant rate of up to 0.8 ± 0.05 (SD) nodules' d -I. When fully developed nodules induced by wild-type or exo mutants were excised and removed from the plastic growth pouch, new nodules were formed on lateral roots. In plants inoculated with exo mutants, a significant increase in both the number of lateral root nodules and the rate of nodule emergence was observed. After nodule excision, the rate of nodulation induced by an exoB mutant on lateral roots doubled (Fig. 1) but never reached the levels obtained when nodules induced by parent R. meliloti 1021 were excised (3.8 ± 0.4 nodules' d -I). Delays in excision had no effect on the rate of nodule formation (Fig. 1). However, the number of nodules formed on lateral roots at the end of the experiment doubled when nodules were excised 12 d post-inoculation but only increased 30 - 40 % when excision was delayed by a
20
A
Lateral roots
I
10
Primary root
30
B
IZ
c:( -I
a..
20
C/)
w
-I
::)
Cl 10
0 Z
20
C
10
DAYS AFTER INOCULATION
Fig. 1: Effect of nodule excision on subsequent nodule emergence. Sets of 47 to 59 plants in groups of five were inoculated with 2 x 106 bacteria/plant of R. meliloti Rm7094 (exoB94:Tn5) 5 d after seed imbibition, and the appearance of nodules on the primary root (0) and on lateral roots (e) was determined at regular intervals. Nodules were either left undisturbed (A) or were excised 12 d (B) or 19 d after inoculation (C). Bars indicate maximum 95 % confidence limits. The results shown are representative of 2 independent experiments. Primary roots reached the bottom of the growth pouch 10 to 12 d after germination.
week. Since no inherent limitation on lateral root nodulation was observed with time (Caetano-Anolles and Gresshoff, 1991), the difference results from the constraints of our experimental design. While very few if any new nodules emerged on the primary root (Fig. 1 Band q, new nodules appeared on the lateral roots particularly in the vicinity of the lateral RT mark made at the time of excision (data not shown). This region of the lateral roots was the one susceptible to infection at the time of nodule removal. Nutman (1952) found that surgical excision of functional nodules or root tips stimulated the formation of new nodules in red clover. This result was interpreted as the ability of established meristematic foci to inhibit new nodule formation and showed that the extent of nodulation was regulated by the host plant. We found that excision of first nodules releases the autoregulatory suppression of nodulation in alfalfa and soybean. Removal of fully developed nodules from alfalfa primary roots caused nodules to emerge almost ex-
Rhizobium meliloti exo mutants control nodulation in alfalfa
elusively around the location of the lateral root tips at the time of nodule excision (Caetano-Anolles and Gresshoff, 1991). In soybean, nodule excision resulted in new nodules emerging on primary and lateral roots in those same regions from where nodules were initially removed and from infections that were already initiated at the time (Caetano-Anolles et al., 1991 b). The different location in nodule emergence indicates the existence of at least two alternative mechanisms that control nodulation in legumes, one favoring multiplicity of infections and the selective arrest of nodule development, the other controlling nodule initiation. Our present studies demonstrate that the excision of bacteria-free nodules induced by exo mutants stimulates the development of additional nodules. Split-root experiments have already shown that these empty nodules elicit the systemic autoregulatory response that controls nodulation (Caetano-Anolles et al., 1990). However, nodule excision experiments in soybean revealed nodulation control mechanisms other than systemic regulatory responses (Caetano-AnolIes et al. 1991 b). It was then important to determine if nodule excision released nodulation control even in the absence of bacterial infection. The observation that nodule removal stimulates nodulation suggests further that organized nodular structures devoid of inter- or intracellular bacteria are active in controlling nodulation. Since approachgrafting of a soybean mutant blocked in nodule development to its parent showed that cortical cell division without infection triggered the systemic regulatory response (CaetanoAnolIes and Gresshoff, 1990) and spontaneous alfalfa nodules formed in the absence of Rhizobium induced autoregulation (Caetano-AnolIes et al., 1991 a), our results suggest that the plant plays a key role in regulation of nodule number. In particular, the existence of an homeostatic control of nodule number as the one reported here can be of relevance in agronomical situations where there is nodule loss by desiccation or defoliation at the time of alfalfa harvesting. Acknowledgements This work was supported by an endowment to the Racheff Chair of Excellence of The University of Tennessee, and the Tennessee Soybean Promotion Board.
767
References BHUVANESWARI, T. v., A. A. BHAGWAT, and W. D. BAUER: Plant Physiol. 68,1144-1149 (1981). CAETANO-ANOLLES, G. and W. D. BAUER: Plant a 175, 546-557 (1988). CAETANO-ANOLLES, G. and P. M. GRESSHOFF: Plant Sci. 71, 69-81 (1990). - - Plant Physiol. 95, 366-373 (1991). CAETANO-ANOLLES, G., A. LAGARES, and W. D. BAUER: Plant Physiol. 92, 368-374 (1990). CAETANO-ANOLLES, G., J. A. JOSHI, and P. M. GRESSHOFF: Planta 183, 77 -82 (1991 a). CAETANO-ANOLLES, G., E. T. PAPAROZZI, and P. M. GRESSHOFF: J. Plant Physiol. 137, 389-396 (1991 b). FINAN, T. M., A. M. HIRSCH, J. A. LEIGH, E. JOHANSEN, G. A. KULDAU, S. DEEGAN, G. C. WALKER, and E. R. SIGNER: Cell 40, 869-877 (1985). HIRSCH, A. M., T. V. BHUVANESWARI, T. G. TORREY, and T. BISSELING: Proc. Natl. Acad. Sci. USA 86,1244-1248 (1989). HIRSCH, A. M., K. J. WILSON, J. D. G. JONES, M. BANG, V. V. WALKER, and F. M. AUSUBEL: J. Bacteriol. 158, 1133-1143 (1984). JENSEN, H. L.: Proc. Linn. Soc. N. S. W. 66, 98-108 (1942). KApP, D., K. NIEHAUS, J. QUANDT, P. MULLER, and A. PUHLER: Plant Cell 2, 139-151 (1990). LEIGH, J. A., E. R. SIGNER, and G. C. WALKER: Proc. Natl. Acad. Sci. USA 82,6231-6235 (1985). LEIGH, J. A., J. W. REED, J. F. HANKS, A. M. HIRSCH, and G. C. WALKER: Cell 51, 579-587 (1987). LEROUGE, P., P. ROCHE, J. PROME, C. FAUCHER, J. VASSE, F. MAILLET, S. CAMUT, F. DE BILLY, D. G. BARKER, J. DENARIE, and G. TRUCHET: In: GRESSHOFF, P. M., E. ROTH, G. STACEY, and W. E. NEWTON (eds.), Nitrogen fixation: achievements and objectives, pages 177 -186. Chapman and Hall, New York (1990). NUTMAN, P. S.: Ann. Bot. N. S. 16, 81-102 (1952). TRUCHET, G., C. ROSENBERG, J. VASSE, J. S. JULLIOT, S. CAMUT, and J. DENARIE: J. Bacteriol. 157, 134-142 (1984). TRUCHET, G., D. G. BARKER, S. CAMUT, F. DE BILLY, J. VASSE, and T. HUGUET: Mol. Gen. Genet. 219, 65-68 (1989). VAN DE WIEL, c., J. H. NORRIS, B. BOCHENEK, R. DICKSTEIN, T. BISSELING,and A. M. HIRSCH: Plant Cell 2, 1009-1017 (1990). WONG, C. H., C. E. PANKHURST, A. KONDOROSI, and W. J. BROUGHTON: J. Cell. BioI. 97, 787 -794 (1983).