CULTIVAR DEVELOPMENT: ACCELERATED INTROGRESSION OF SYNTHETIC HEXAPLOID DERIVED DIVERSITY INTO AN APPLIED HARD WINTER WHEAT BREEDING PROGRAM

Our previous work has focused on signaling and gene regulation in two model bacteria that undergo sporulation in response to starvation. Sporulation is an adaptive response that allows bacteria to survive in the absence of food by becoming dormant. Cells halt their metabolism and build protective layers to withstand harsh environmental conditions such as dryness, heat, and sunlight. The two bacteria we study undergo very different sporulation processes.Bacillus subtilis is partitioned into two compartments, the mother cell and the forespore, each of which expresses distinct sets of genes in an ordered temporal fashion, under the control of different sigma (sig) subunits of RNA polymerase.Our previous work on B. subtilis focused on gene regulation in the mother cell. While investigating genes that are expressed during the later stages of sporulation under the control of SigK RNA polymerase, we discovered that SigK is first made as an inactive precursor (Pro-sigK) and that processing to the active form in the mother cell depends on a signal from the forespore. We characterized this novel signal transduction pathway. We showed that SpoIVFB is the protease that processes Pro-sigK. It is an intramembrane metalloprotease (IMMP) that cleaves Pro-sigK within or adjacent to the outer forespore membrane. Homologs of SpoIVFB are present in nearly all organisms studied. We are also investigating RasP, a second IMMP of B. subtilis, which is representative of a type that is even more broadly conserved than SpoIVFB. Bacterial IMMPs are known to play important roles during stress responses, mating, polar morphogenesis, cell division, and infection. Understanding how IMMPs function in bacteria could lead to the development of new antibiotics. In eukaryotes, IMMPs cleave transcription factors that regulate lipid metabolism in animals, chloroplast development in plants, and responses to endoplasmic reticulum stress in both animals and plants. Knowledge about bacterial IMMPs will facilitate studies of eukaryotic IMMPs, which could lead to the development of novel therapeutics and growth modulators. We expect our work on SpoIVFB and RasP to provide new insights into how IMMPs cleave their substrates and how IMMP activity can be modulated. These insights could guide efforts to develop IMMP modulators. Our work on RasP may also reveal a new biological function(s) of IMMPs by identifying a novel substrate(s).The objectives of our B. subtilis work are:1. Test the model that the ATP level rises in the mother cell compartment after the completion of forespore engulfment and that ATP binding to the CBS domain induces a conformational change in SpoIVFB that positions Pro-sK for cleavage.2. Determine how BofA and SpoIVFA inhibit SpoIVFB.3. Test whether RasP cleaves FtsL without a prior cleavage and identify a novel substrate(s) of RasP.The second bacterium we study, Myxococcus xanthus, undergoes a multicellular developmental process. When starved, cells change their gene expression and metabolism, send signals to each other, move in streams to construct multicellular mounds (nascent fruiting bodies), and some of the rod-shaped cells differentiate into round, dormant spores. Other cells remain outside of fruiting bodies as peripheral rods and the majority of cells undergo lysis.Our previous work focused on understanding how genes are regulated in response to C-signaling during fruiting body development. C-signaling involves CsgA, a protein that becomes associated with the cell surface and mediates short-range signaling between cells. C-signaling regulates not only gene expression, it also coordinates streaming and mound formation with spore differentiation, but the mechanisms driving these emergent behaviors are not fully known. We discovered that several C-signal-dependent promoters are under combinatorial control of two transcription factors, MrpC and FruA, but genes important for emergent behaviors remain to be identified.We expect the project to provide deep insight into emergent properties found commonly in development (assembly of multicellular structures and cell fate determination) using a highly genetically tractable system. This knowledge has potential to transform thinking about emergent behaviors and the evolution of multicellularity. The knowledge gained and methods developed will benefit efforts to understand the alternative M. xanthus cell fates of lysis and peripheral rod formation, and will catalyze research on microbial communities (biofilms, microbiomes). Microbiomes impact all organisms and the ecosystems they inhabit. Our understanding of how cells in microbiomes interact with each other and their environment is limited, impeding our ability to intervene for the benefit of society (e.g., to manipulate microbiomes for pollution and climate control, and for increased bioenergy and food production). Knowledge from the project is expected to help guide such efforts.The objectives of our M. xanthus work are:1. Test whether expression of C-signal-dependent genes increases during streaming and mound formation, and identify genes important for those emergent behaviors.2. Test whether C-signaling triggers a new pattern of gene expression at the onset of the rod-to-spore transition and identify genes important for initiating cellular shape change.3. Determine the mechanisms of C-signal transmission and FruA activation that drive threshold-dependent gene expression and successive emergent behaviors.