Requirement for rhizobial production of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) for infection of bean

Exogenous application of the purine precursor 5-aminoimidazole-4-carboxamide (AICA) riboside restores infection and enhances the development of bean (Phaseolus vulgaris) root nodules elicited by Rhizobium etli purine auxotrophs. The uniqueness of AICA riboside as an effector was shown by testing varying concentrations of AICA riboside and purines for this effect, and by examining several mutants defective in various pathways of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) synthesis. The maximum effect on nodule development was achieved at 0.1 mM AICA riboside. The purines adenine, adenosine, or hypoxanthine did not enhance nodule development at any concentration, but at very high concentrations (1.0 mM), inosine did promote infection. Studies with a double mutant indicated that the histidine biosynthetic pathway (in which AICAR is a byproduct) was required for the effect of inosine, but not for the effect of AICA riboside. A mutant resistant to pyrazofurin, a toxic AICA riboside analog, was isolated and found to be defective in conversion of AICA riboside to AICAR. Transfer of a purF::Tn5 mutation into this strain yielded a purine auxotroph that did not grow with AICA riboside as a purine source. AICA riboside failed to promote infection by this double mutant, indicating that AICA riboside does not act directly upon the plant, but rather must be converted to AICAR by rhizobia in order to be effective. In summary, these results indicate that infection of bean by R. etli requires rhizobial production of AICAR, whether via purine biosynthesis, histidine biosynthesis, or conversion from AICA riboside.