The Addition of 5-Aminoimidazole-4-Carboxamide-Riboside to Nodulation-Defective Purine Auxotrophs of NGR234 Restores Bacterial Growth but Leads to Novel Root Outgrowths on Siratro. Steven P. Djordjevic. Plant Microbe Interaction Group, Research School of Biological Sciences, The Australian National University, P.O. Box 475, Canberra City, Australian Capital Territory 2601, Australia. Jeremy J.Weinman, John W. Redmond, Michael A. Djordjevic, and Barry G. Rolfe Plant Microbe Interaction Group, Research School of Biological Sciences, The Australian National University, P.O. Box 475, Canberra City, Australian Capital Territory 2601, Australia. MPMI 9:114-124. Accepted 13 October 1995, Copyright 1996 The American Phytopathological Society.

The transposon Tn5-induced purine auxotrophs of NGR234 (ANU2861 and ANU2866) are defective for nodule formation on a wide variety of legumes and the non-legume Parasponia. Complementation studies and DNA sequence analysis of regions, on either side of the insertion of Tn5 in ANU2861, revealed two open reading frames with strong sequence homology to the purM and purN genes of Escherichia coli. Strain ANU2861 contained a single copy of Tn5 inserted into the 3' region of the putative purM gene. On Macroptilium atropurpureum (siratro) ANU2861 rapidly induces a reaction analogous to a hypersensitive response at the site of infection (S. P. Djordjevic, R. W. Ridge, H. Chen, J. W. Redmond, M. Batley, and B. G. Rolfe, 1988, J. Bacteriol. 170:1848-1857). However, when the plant growth medium was supplemented with 0.1 mM 5-aminoimidazole-4-carboxamide riboside (AICA-riboside), strains ANU2861 and ANU2866 induced the formation on siratro of root outgrowths that resembled Fix' nodules. These outgrowths do not contain bacteria, lack infection threads, and possess centrally located vascular bundles in contrast to the peripheral location in functional nodules induced by wild-type strain NGR234. When mutant ANU2861 was spot inoculated onto the roots of siratro, or coinoculated onto the roots with the ExoY mutant ANU2811, a thick and short root (Tsr) phenotype was induced. The roots of siratro plants inoculated with parent strain ANU280 do not induce a Tsr-like phenotype. Complementation of ANU2866 for nodulation and prototrophic growth was achieved by a cosmid containing purY, purQ, and purL genes. The examination of ANU2861 and ANU2866 lipopolysaccharide (LPS) showed that only ANU2861 possessed a defective LPS that was corrected by the addition of AICA-riboside to the growth medium. These results indicated that both mutants are defective in the purine biosynthetic pathway and that metabolic flow through this pathway is essential for nodule morphogenesis.