A comprehensive systems-level investigation of the responses of Setaria viridis to growth promoting associations involving

While nitrogen management is an important agronomic trait in any cropping system, it is of particular concern for future bioenergy crops that will be grown on sub-optimal soils lacking this essential resource for sustainable plant growth. Use of N fertilizers will improve upon this, but significantly increase the net cost for energy derived from bioenergy fuels. However, an increasing number of reports have appeared documenting plant growth enhancement by nitrogen fixing plant growth promoting rhizobacteria (PGPR), which has increased the acceptance of their commercial use. Even so, most aspects of these unique plant-microorganism associations have been little studied at a systems-level and from a detailed mechanistic perspective. Knowledge gained from more intensive research on PGPR and their effects should increase their utility and field application in future bioenergy cropping systems. We believe what is lacking is a genetically tractable, model system that can be employed to make more rapid progress to understand PGPR function. Therefore, we propose to study Setaria viridis as a model grass system for exploring mechanisms underpinning early bacterial association by Herbaspirillum seropedicae and Azospirillum lipoferum with their hosts. Using a systems biology approach, S. viridis plants will be extensively studied at transcriptomic and proteomic levels leveraging available genomes, and at microscopic, physiological and biochemical levels using compositional and flux analysis, the later leveraging stable and radioactive tracers (11/13C and 13N) to measure changes in dynamic allocation patterns and metabolic partitioning of carbon and nitrogen resources. This project reflects a joint effort between BNL and the University of Missouri where team members already have one proposal submitted to the USDA-DOE Feedstock Program (FOA No: DE-FOA-0000770) and will be leveraging re-purposed DOE/BER funds at BNL (FWP BO158) to jump start studies this summer. If this EMSL project is approved, the expectation is that advanced proteomic and metabolomic expertise and instrumentation available at EMSL will be leveraged providing an unprecedented thorough analysis from a genomic scale to the whole-plant scale enabling us to determine how these bacteria establish themselves as symbionts with their host during early stages of infection, and to establish a fundamental understanding of the mechanism for their promotion of plant growth. The specific project objectives are: (1) Characterize host growth responses to bacterial infection using both wild-type and mutant H. seropedicae and A. lipoferum strains that will optically report on root colonization; (2) Characterize host transcriptional and proteomic responses to infection by wild-type H. seropedicae and A. lipoferum strains leveraging colonization data from Obj. 1 to target sites for tissue collection; and (3) Characterize host physiological and metabolic responses to infection by wild-type H. seropedicae and A. lipoferum strains utilizing isotopic labeling.