Legumes contribute 1/3 of humankind's protein intake, a fact that is directly related to their unusual capacity to access atmospheric nitrogen through symbiosis with nitrogen-fixing bacteria. Biological nitrogen fixation also makes legumes key components of both natural and agricultural systems because they return vital nitrogen to the environment. This project will extend existing knowledge of how legumes and their symbiotic bacteria establish this unique partnership, in particular by bridging traditional genetic methodologies with specialized tools that the investigators have developed for protein biochemistry and cell biology. The outcome of these studies will be increased understanding of a critical phase in symbiotic development, namely the period during which legumes select efficient symbiotic bacteria from the soil environment. In particular, the proposed research will identify new genes and proteins that underlie symbiotic nitrogen fixation. Knowledge of symbiotic nitrogen fixation has great relevance to agricultural systems, because US agricultural is highly dependent on input nitrogen, which for non-legumes involves consumption of fossil fuels to drive conversion at atmospheric nitrogen (N2) to fertilizer nitrogen. The fact that legumes produce fertilizer nitrogen directly, using solar energy obtained through photosynthesis, makes them key components of cost-effective, carbon-neutral, sustainable agricultural systems. Increased understanding of symbiotic development will enhance our ability to constructively manipulate this vital biological process. The anticipated broader impacts of this work include training of graduate students, and students will participate in an active international collaboration with scientists in the Republic of Korea through the Korean Rural Development Administration.
Symbiotic nitrogen fixation has global importance on par with photosynthesis. In the developed world fossil fuels drive industrial conversion of atmospheric di-nitrogen to ammonia at great cost, while in the developing world legumes are underutilized as a source of green nitrogen. Increased understanding of symbiotic nitrogen fixation, including control of symbiotic initiation, has the potential to benefit both traditional agricultural systems and bio-energy crops. The rhizobium-legume symbiosis is also a paradigm for plant-bacterial interactions and a model system for understanding ligand-stimulated development in plants. At the outset of symbiotic development, symbiotic bacteria deliver a host-specific "Nod factor" ligand to the plant root, which triggers a cascade of cellular and developmental responses that culminate in formation of a symbiotic root organ. Under this NSF award, we have used tools of protein biochemistry, cell biology and genomics, to identify novel genes an proteins involved in the initial phase of symbiotic development. We have used a co-purification strategy to identify novel host proteins that interact with a symbiotic receptor kinase, DMI2. We have determined that DMI2 localizes to the host cell plasma membrane in punctate domains. Among the co-purifying proteins is a homolog of mammalian "band 7/SPFH" proteins, which we have named MtHIR. In plants, HIR homologs are implicated in defense responses to pathogens, while in animal systems band7/SPFH proteins are characterized as membrane micro-domain associated proteins. By analogy, we hypothesize that MtHIR will localize to membrane domains that also contain DMI2. We have validated the interaction between MtHIR and DMI2 using yeast-2-hybrid assays, and we have shown that transcription of DMI2 is specifically activated by Nod factor in a manner that also requires perception of the plant hormone ethylene. Thus, HIR is subject to positive regulation by both a plant and a bacterial signal. Using a reverse genetic screen, known as TILLING, we identified 15 EMS-induced alleles of the candidate HIR protein. Six alleles are predicted to impact gene function, of which one is a stop codon (putative loss of function). Functional redundancy from close paralogs may require RNAi silencing of multiple family members to assess gene function and we intend to use the TILLING stop allele as the basis for such studies. As a complement to proteomic-based discovery of interacting proteins, we have used RNAseq next-generation sequencing techniques to analyze the transcriptional changes in Medicago truncatula roots at very early symbiotic stages. Our two-fold aim was to identify symbiosis-specific genes whose transcription is activated by Nod factor, and to characterize the ethylene biosynthesis and signaling pathways in M. truncatula in the context of early symbiotic development. The outcome is a deep analysis of the early transcriptional changes occurring in M. truncatula hours after the inoculation of rhizobium, providing detailed digital time courses of gene expression and highlighting numerous novel genes co-expressed with known markers of symbiosis. Following normalization and imposition of a conservative 0.001 false discovery rate cutoff we circumscribed 10,985 genes, among which 3,631 were differentially expressed between A17 and nfp, 1,996 between A17 and lyk3, and the vast majority, 8,760, between A17 and skl. Many of these genes are strong candidates for novel components of NF-signaling and early symbiotic developmental processes.
