Intracellular Accommodation of Mycorrhiza and Rhizobia by Plants: Molecular Mechanisms and Evolution

All plants form intimate associations with microbes, such as bacteria and fungi. These associations, commonly called symbioses, can be either beneficial or harmful to the plant. The proposed research is to learn more about the function of a gene that is required for two such symbioses that have great agricultural importance. More than 75 percent of plants, including most important crops, form a beneficial symbiosis with a soil fungi call arbuscular mycorrhizal fungi (AMF). This symbiosis evolved more than 400 million years ago about the time plants began to colonize land. During AMF symbiosis a plant acquires important nutrients from the fungus, including phosphate, nitrogen and water, in exchange for sugar produced by the plant during photosynthesis. The AMF effectively extend the reach of the plant root system but rely on a supply of plant sugar for survival. The second symbiosis selected for study in this proposed research is the legume-rhizobia symbiosis. Legumes are the third largest plant family with more than 18,000 members. Legume plants seeds are particularly protein rich and provide about 25 percent of protein in the human diet. Legume forage crops like alfalfa are prized for their high protein content in their leaves, which makes them ideal food for livestock. The primary reason legumes offer such nitrogen-rich seeds and leaves is the legume-rhizobia symbiosis. In this symbiosis the rhizobial soil bacteria invade the legume roots. Once inside the plant cells, they begin converting nitrogen gas from the surrounding atmosphere into ammonia, a form of nitrogen that plants can use. This type of nitrogen is called 'fixed' nitrogen and the process is called nitrogen fixation. This adaptation means that legumes can grow in soils that are deficient in fixed nitrogen without the addition of nitrogen fertilizer. Also when these legumes die, they leave behind nitrogen that can be used by non-legume crops, a feature that is highly valued in modern agriculture. In both the AMF and rhizobial symbiosis, the microbes enter the plant's cells by a mechanism that is under the control of the plant. I have recently discovered a plant gene that produces a protein necessary for both AMF and rhizobial entry to occur. We will study this protein to better understand how the plant allows these helpful microbes into its cells. A part of the research is directed at examining where the new protein is found within the cell, what it and how it might interact with other plant proteins. In addition, an approach will be used that will help identify other proteins that could potentially be involved. In addition to studies at the cellular level, the research also will also examine another possible role for this protein. Many harmful disease-causing fungi invade plant cells, and it is possible they gain entry into the host plant by turning on the same genes used by the rhizobia and AMF symbioses. By examining these diseases for the involvement of same protein found in AMF and rhizobial symbioses, we can test this idea, which could potentially reveal key insights into plant diseases. One interesting aspect of the research concerns the lupin family of legumes which does not form AMF symbiosis and also has an unusual form of rhizobial symbiosis that does not use the typical entry into the plant cell. Figuring out whether lupins have this gene may help explain why they are different and also will shed light on how these symbiotic associations evolve.

The two most agriculturally important beneficial associations of plants with microbes are the arbuscular mycorrhizal fungi (AMF) and rhizobial symbioses. Over 75% of plants, including most important crops, form a beneficial symbiosis with arbuscular mycorrhizal fungi (AMF) which provides the plant with important nutrients such as phosphate, nitrogen and water. The majority of the 18 000 species of legume family of plants engage in a nitrogen fixing symbiosis with plant soil bacteria called rhizobia, including many important crops species. In both the AMF and rhizobial symbioses the microbes enter the plant cells, forming membrane bound compartments within the cells where nutrients are exchanged. This plant-controlled access to the cell is termed intracellular accommodation. In this fellowship I will take genetic and cell biological approaches to identify the components and mechanisms that control this phenomena. My current research has involved the identification of a novel plant gene, Hermes, which is essential for both rhizobial and AMF invasion. A part of the proposed research is directed at identifying where this protein is found within the cell, what it is doing and how it might interact with other plant proteins. A collection of mutants will be identified that are defective in rhizobial and AMF entry using forward and reverse genetic approaches. I will use the technique of laser capture microdissection in combination with deep sequencing to identify genes that are specifically expressed within the tissues accommodating rhizobia and AMF. Another aspect of the project will explore whether Hermes is involved in the intracellular accommodation of harmful disease-causing fungi which form feeding structures analogous to arbuscules. Finally evolutionary aspects of the Hermes protein will be explored by testing to whether Hermes is present in lupins, a plant family that has lost the ability to form AMF associations and rhizobial infection threads.