Purine auxotrophs of various Rhizobium species are symbiotically defective, usually unable to initiate or complete the infection process. Earlier studies demonstrated that, in the Rhizobium etli-bean symbiosis, infection by purine auxotrophs is partially restored by supplementation of the plant medium with 5-aminoimidazole-4-carboxamide (AICA) riboside, the unphosphorylated form of the purine biosynthetic intermediate AICAR. The addition of purine to the root environment does not have this effect. In this study, purine auxotrophs of Rhizobium fredii HH303 and Rhizobium keguminosarum 128C56 (bv. viciae) were examined.Nutritional and genetic characterization indicated that each mutant was blocked in purine biosynthesis prior to the production of AICAR. R. fredii HH303 and R. leguminosarum 128C56 appeared to be deficient in AICA riboside transport and/or conversion into AICAR, and the auxotrophs derived from them grew very poorly with AICA riboside as a purine source. All of the auxotrophs elicited poorly developed, uninfected nodules on their appropriate hosts. On peas, addition of AICA riboside or purine to the root environment led to enhanced nodulation; however, infection threads were observed only in the presence of AICA riboside. On soybeans, only AICA riboside was effective in enhancing nodulation and promoting infection. Although AICA riboside supplementation of the auxotrophs led to infection thread development on both hosts, the numbers of bacteria recovered from the nodules were still 2 or more orders of magnitude lower than in fully developed nodules populated by wild-type bacteria. The ability of AICA riboside to promote infection by purine auxotrophs, despite serving as a very poor purine source for these strains, supports the hypothesis that AICAR plays a role in infection other than merely promoting bacterial growth.Root nodules of leguminous plants develop from cell division foci triggered within young root cortical layers by signals from bacteria of the genera Rhizobium, Bradyrhizobium, and Azorhizobium. The bacteria penetrate from the root epidermis to the interior of the developing nodule by means of an infection thread, composed of a growing linear bacterial colony and the surrounding tubular cell wall structure produced by the plant. Eventually, numerous plant cells in the nodule interior become infected by endocytosis of the bacteria from unwalled tips of the infection thread (17). These released bacteria then differentiate into bacteroids that commence nitrogen fixation.The process of infection thread formation remains obscure, especially at the molecular level. Very little is known about the contribution of the microsymbiont, although it is clear that certain deficiencies of the bacteria can prevent or severely limit infection. Mutations in crucial bacterial nod genes prevent infection, but since they also prevent (5,7,12,22,25,32) and auxotrophs (2,6,9,10,16,18,23,(26)(27)(28)30). Perhaps the most universally deleterious such defect is purine auxotrophy.Purine auxotrophs (Pur-) of Rhiz...

Purine auxotrophs of Rhizobium leguminosarum biovar phaseoli CFN42 elicit uninfected pseudonodules on bean (Phaseolus vulgaris L.). Addition of 4-aminoimidazole-5-carboxamide (AICA) riboside to the root medium during incubation of the plant with these mutants leads to enhanced nodule development, although nitrogenase activity is not detected. Nodules elicited in this manner had infection threads and anatomical features characteristic of normal nodules, such as peripheral vasculature rather than the central vasculature of the pseudonodules that were elicited without AICA riboside supplementation. Although 105 to 106 bacteria could be recovered from these nodules after full development, bacteria were not observed in the interior nodule cells. Instead, large cells with extensive internal membranes were present. Approximately 5% of the normal amount of leghemoglobin and 10% of the normal amount of uricase were detected in these nodules. To promote the development of true nodules rather than pseudonodules, AICA riboside was required no later than the second day through no more than the sixth day following inoculation. After this period, removal of AICA riboside from the root medium did not prevent the formation of true nodules. This observation suggests that there is a critical stage of infection, reached before nodule emergence, at which development becomes committed to forming a true nodule rather than a pseudonodule.