EXPRESSION PROFILING THE Melilotus alba-Sinorhizobium meliloti NITROGEN FIXING SYMBIOSIS.

¹ Dept. of Molecular Cell & Developmental Biology, UCLA, Los Angeles, CA 90095 USA

² Dept. of Chemistry and BioScience, Kagoshima University, Kagoshima 890-0065, Japan

Melilotus alba (white sweetclover) is a small, diploid legume with a short life cycle, ca. 3 months. It forms a nitrogen-fixing symbiosis with Sinorhizobium meliloti, whose genome has been sequenced. The genome size of M. alba (1254 Mbp per haploid genome) is comparable to that of tomato (ca. 1000 Mbp) and is approximately 6 times the size of Arabidopsis. Mutagenesis of line U389 has yielded numerous mutants. Of the symbiotic mutants, sym3 lacks root hair deformation, root cortical cell division, and infection thread formation, placing the mutation at an early stage of interaction with S. meliloti. We are studying genes involved in this early stage and have employed cDNA-AFLP to analyze the expression profile. This involves the comparison of genes expressed between wildtype U389 root tips inoculated with S. meliloti strain GMI6032, a Nod factor overproducing strain, and mock-inoculated U389 root tips. Another comparison was made between GMI6032-inoculated U389 and sym3 root tips. We have found that genes involved in the defense response, for transcription factors, and for components of signaling cascades appear to be important for establishment of this symbiosis. To study these genes further, we are developing Agrobacterium-mediated plant transformation techniques. Several methods have been explored: shoot organogenesis, radicle excision, and vacuum infiltration of seeds, flowers, and roots. Addition of antioxidants and plant defense moderators in the media play a critical role in the efficiency of plant transformation. A set of conditions for hairy root transformation has been developed for increasing the transformation efficiency of M. alba.