Integration into rice of a soybean apyrase gene proposed to play a central role in nodulation

Perception of rhizobial, lipo-chito-oligosaccharide nodulation (nod) signals by the legume host is a key step in symbiotic development. Recently, Etzler et a!. (1999) reported the isolation of an apyrase (i.e., nucleotide phosphohydrolase) from the legume Dolichos biflorus that had the ability to bind to rhizobial Nod signals. Therefore, this protein is a possible candidate for a Nod signal receptor. An ortholog (GS5O) of the D. biflorus apyrase was isolated from soybean (Glycine soja)(Stacey et a!. 1999). The sequence of this gene clearly indicates that it falls into the apyrase family and in vitro expression of this protein demonstrates that it possesses nucleotide phosphohydrolase activity. We have introduced a plasmid harboring this nod factor binding protein gene (Fig. 1), driven by a constitutive CaM V35S promoter, into rice cells using the biolistic method. Isolated immature embryos from the japonica cultivar, Taipei 309, were used as explants. Southern blot analyses of the T0 and T, generation plants showed several independent transformation events, stable integration and inheritance of the transgene (Fig.2). So far , out of nearly 100 putative transgenics obtained, 13 plants showed integration of the expected 1.5 Kb fragment of the gene with ten independent lines. Molecular analysis also revealed that the promoter region has been integrated along with the gene.

Recent findings show that rice genome does possess varied levels of homology with several nodulin genes of legumes, including ENOD4O (Kouchi eta!. 1999). We are currently working to develop a system by which we can demonstrate the nod signal binding activity of GS5O and its involvement in nodulation. We also plan to test whether the introduction of this gene into rice can confer the ability to perceive the lipo-chitin nod signal. The hope is that this basic research will extend our understanding of the inherent inability of rice to respond to rhizobial inoculation. Our hypothesis is that rice possesses some (e.g., nodulins) but not all of the traits found in legumes that are important for nodulation. If this hypothesis is correct, then reconstituting the nod signal recognition pathway in rice could allow this plant to interact more intimately with rhizobia. Furthermore, this finding would provide further support for the idea that �legume plants acquired the ability to form symbiotic nitrogen fixing nodules by recruiting genes that have common functions in all plants� (Mylona et al. 1995, Carol et al. 1996). Can the counterparts of these genes in rice be re-recruited to form functional nodules? Experiments to study expression of the GS5O transgene and correlation with phenotypes are underway.

Acknowledgement

Financial supports from the Rockefeller Foundation, NY, DANIDA, Denmark are gratefully appreciated. Worked performed in the laboratory of GS was funded, in part, by a grant from the US Department of Energy, DE-FGO2-97ER-20260.

References

Etzler, M.E., G. Kalsi, N.N. Ewing, N.J. Roberts, R.B. Day and J.B. Murphy, 1999. A nod factor binding Lectin with apyrase activity from legume mets. PNAS USA, 96: 5856-5861.