A Ca2+/calmodulin-dependent protein kinase (CCaMK) has been implicated as a key player in the signal transduction cascade that follows perception of nitrogen fixing bacterial symbionts by plant roots and leads to the establishment of nitrogen-fixing root nodules. This project seeks to determine the role of this kinase in roots as signals from potential symbionts are received and transduced to downstream effectors involved in nodule formation. CCaMK, a multi-functional protein, is characterized by the presence of a kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like Ca2+-binding domain in a single polypeptide. CCaMK undergoes two steps of Ca2+ regulation, first directly through its Ca2+-binding domain and then indirectly through its Ca2+/calmodulin-binding domain. Recent investigations have revealed that CCaMK plays critical roles in both bacterial and fungal symbioses. During bacterial interactions which lead to nitrogen fixation, CCaMK acts immediately downstream of Ca2+ oscillations triggered in root hair cells by the bacterially produced signaling molecule, Nod factor. The immediate goal of this investigation is to study how the structural changes of CCaMK control bacterial invasion and the resulting symbiotic relationship. Furthermore, the project will also seek to identify and characterize CCaMK's potential downstream interaction partner(s). Genetic studies indicate that CCaMK is at a critical juncture of a branch point in the signaling pathway which mediates the interaction of plants with their symbionts: one branch leads to nodulation and the other to mycorrhizal infection. Hence, this project may lead to new insights into fungal as well as bacterial symbioses. This project will use genetic, biochemical and cellular approaches to address a seminal question in the area of plant:microbe interactions. In addition to its agricultural and ecological significance the broader impacts of the project include training undergraduates, graduate students and postdocs. Training of minorities and women who are interested in science will be emphasized. On-site participants will experience the atmosphere of an active biotechnology research laboratory. To reach a wider audience, the WSU team will also use the Washington State Higher Education Telecommunication System and organize onsite workshops.
The Rhizobium-legume symbiosis fixes about the same amount of nitrogen worldwide as the chemical fertilizer industry produces. Ever since Beijerinck’s demonstration that soil bacteria, rhizobium, cause nodule formation, the mechanisms of nitrogen fixation have attracted serious study due to the major impact on energy-saving, agriculture and eco-systems. The symbiotic relationship between the plant and the bacteria is a complex process tightly regulated by both partners. For example, plants allow only specific bacteria to infect the roots and control the nodule formation during the course of symbiosis. The ability of bacteria to reduce atmospheric nitrogen and to supply it to plants depends on the quality and quantity of carbon and nitrogen source provided by the plant. Intensive farming depends heavily on inorganic fertilizers that are often used to provide nutrients, particularly nitrogen, that are critical for plant growth. The production of nitrogen fertilizers requires a lot of energy and is estimated to constitute approximately fifty percent of the fossil fuel usage of the modern agricultural process. The heavy dependence on inorganic fertilizers in modern farming causes environmental problems associated with leeching into streams, lakes and underground water reserves. The nitrogen leaches from farm fields into groundwater and streams, at times reaching high enough levels to kill nearly everything in the water. It is estimated that thousands of square miles of the Gulf of Mexico are now a "dead zone" due to the 1.5 million metric tons of nitrogen fertilizer that are washed every year from mid-American farms into the Mississippi River and ultimately into the ocean. Hence, there is a great deal of interest in understanding the mechanisms involved in symbiotic nitrogen fixation and to create crop plants that can fix their own nitrogen. This laboratory has a long history of working on calcium/calmodulin-mediated signaling in plants. In 1995, this laboratory published data that described the cloning and characterization of a chimeric calcium/calmodulin-dependent protein kinase gene (CCaMK) with a neural visinin-like calcium-binding domain (Proc. Natl. Acad. Sci. 92: 4897-4901, 1995). Since 2005, the PI’s team published a series of reports that documented critical roles of calcium/calmodulin-mediated signaling in plant growth and plant-microbe interactions (Nature, 437:741-745, 2005; Nature, 441:1149-1152, 2006; and Nature, 457:1154-1158, 2009; highlighted in Cell 136:193-195, 2009). It is becoming clear that the CCaMK gene plays a central role in both bacterial and fungal symbioses, a topic that is of global agricultural and ecological importance. During bacterial interactions which lead to nitrogen fixation, CCaMK acts immediately downstream of Ca2+ oscillations triggered in root hair cells by the bacterially produced signaling molecule, Nod factor. Our collaboration with Dr. Giles Oldroyd of the John Innes Centre, UK has produced a major breakthrough demonstrating that point mutations and deletions of CCaMK can lead to autoactivation and spontaneous nodulation (Nature 441:1149-1152, 2006). During this funding period, this laboratory carried out comprehensive structure:function dissections of CCaMK to gain further insight into how CCaMK activity is regulated, and what is the impact of these regulatory events on the symbiotic relationship with bacterial or fungal symbionts. Our results revealed that the CCaMK activity is tightly controlled by an auto-inhibitory mechanism in which a particular portion of the CCaMK folds back to its kinase catalytic site to prevent its access to the downstream targets. A pair of interacting amino acids in the kinase and regulatory domains, which are required for the auto-inhibition, was identified and their interaction was confirmed. Another important observation was the discovery of a new regulatory mechanism. Two novel phosphorylation sites were identified in the kinase domain of CCaMK and in the region which interacts with calmodulin. These novel mechanisms enable CCaMK to be turned on and off by calcium and calcium-loaded calmodulin, providing the molecular mechanism for CCaMK to be an "intelligent" calcium signal processor, capable of making a decision to start nodulation or the mycorrhization pathway based on the type of calcium signal it perceives. These findings should help to improve our knowledge base and to control symbioses in plants. The PI and Co-PI integrated research findings into the broader educational arena. We trained undergraduates, graduate students and postdoctoral researchers. Training of minorities and women who were interested in science was emphasized. During this funding cycle, two students completed their Ph.D. degrees. Two postdoctoral researchers and six undergraduate students were also trained. The PI was involved in various outreach activities involving high school and college students. In addition, the PI was also involved in developing an NSF-REU site proposal to train more high school and college students in genomics and biotechnology.
