Genetic Network Regulating the Early Symbiotic Responses in Legumes

Formation of symbiotic nodules in legumes requires plant recognition of rhizobial signal molecules termed Nod factors (NF). Over the past decade, numerous plant genes responsible for NF signal perception and transduction have been identified and cloned. Among the encoded proteins are a series of receptor like kinases that perceive and transduce NF perception, ion channels and kinases that modulate and perceive changes to nuclear calcium concentrations, and the transcription factors that reprogram root cell gene expression. Little is known about the early transcriptional networks that activated by NF perception. To elucidate the early transcriptional networks, we performed RNAseq analyses on wild type (A17), nod- mutants (nfp and lyk3), and the nod++ ethylene insensitive mutant (skl). Our analyses revealed several early NF induced transcripts that are upregulated in A17 but not in the nfp ad lyk3 mutants. Most of these genes are massively up regulated in the ethylene insensitive mutant skl, consistent with the negative regulation of NF signaling by ethylene. Among the set of early NF-induced genes, we identified several genes that encode putative new signal transduction proteins, including kinases and transcription factors. We have used yeast two-hybrid assays to determine if these putative NF signaling proteins interact with the known symbiotic receptor kinases. We have identified a transcription factor and a novel receptor kinase that interact with the symbiotic receptor kinase DMI2 in yeast. We are co-expressing these genes as FLAG fusions with DMI2:HA to assess their interaction in vivo by co-immunoprecipitation.