The expression of the Rhizobium tropici guaB gene is required in the early stages of bean nodulation

COLLAVINO, M.M., GRASSO, D.H., RICILLO, P.M. AND AGUILAR O.M.

Ciudad Miguel Pereira, Brasil.

Congreso; XXII Reunión Latinoamericana de Rhizobiología. I Reunión Brasilera de Fijación de Nitrógeno; 2004

The expression of the Rhizobium tropici guaB gene is required in the early stages of bean nodulation.  Mónica Collavino1,  Daniel H. Grasso 2, Pablo M. Riccillo1,  and O. Mario Aguilar1.  1IBBM-Facultad de Ciencias Exactas, UNLP. Calle  47 y 115,  La Plata, Argentina; 2 Inst. de Suelo, INTA Castelar, Argentina.   We have previously reported that a guaB mutant strain of Rhizobium tropici is defective in symbiosis with Phaseolus vulgaris, forming nodules that lack intracellular bacteroids. The guaB gene encodes for the enzyme IMPDh of the guanine biosynthetic pathway. In this study, we placed the guaB gene under the control of the symbiotic promoters, nodA, bacA and nifH, which activate at different stages of the nodulation in order to assess the timing of guaB requirement for successful nodulation. Additionally, a promoterless gus gene sequence was cloned downstream guaB in order to monitor the transcriptional activity of guaB gene during nodule formation. These constructs carried by the replicative vector plasmid pSUP104 were transferred into the guaB mutant R. tropici CIAT899-10.T. The strains derivatives of R. tropici CIAT899-10.T carrying the three fusions, were tested for their symbiotic phenotype. This was done by observing the appearance of beans nodules, the measuring of nitrogenase activity, and by histological examination. The expression of guaB under the control of the nod promoter, restores the symbiotic wild type phenotype of the mutant strain CIAT899-10.T. In contrast, the nodules formed by the mutant strain carrying the PnifH-guaB fusion, were identical to the nodules of the mutant strain. However, guaB expression under the control of the bacA promoter, supports partial complementation of the defective phenotype, providing limited ability for the mutant to invade only certain zones of the nodule. In addition, we assesed the expression patterns of the symbiotics promotors during the different stages of nodulation, and found that they expressed as previously reported, for the expression of genes which these promoters control. In conclusion, our results indicated that the expression of rhizobial guaB is required from early steps of nodulation, and that the timing of this expression is important for proper nodule development. Additionally, we  examined the role of the guaB gene in the alfalfa nodulation. Thus, a guaB defective mutant derived from Sinorhizobium meliloti 1021 was obtained by fragment specific mutagenesis. We found that mutations in the guaB of S. meliloti do not affect alfalfa nodulation nor nitrogen fixation capacity. Supported by ANPCyT PICT No.7072/99