The nitrogen-fixing mutualism between rhizobia and legumes is one of the best-studied interactions between plants and bacteria. Two major groups of rhizobia, 1) alpha-proteobacteria, i.e. the family Rhizobiaceae (alpha-rhizobia), and 2) certain beta-proteobacteria, Burkholderia and Cupriavidus (beta-rhizobia), inhabit legume root nodules, converting inert atmospheric nitrogen into ammonia, which is assimilated by plants into proteins and nucleic acids. Surprisingly, little is known about the interactions with beta-rhizobia, especially for the Caesalpinioid legumes, the most basal of the three sub-classes of the legume family. Although more than 80% of Mimosoid and Papilionoid legumes nodulate, less than 30% of the Caesalpinioid species form nodules. Thus, they represent a transition from non-nodulating to nodulating legumes and can help tease out the evolutionary origins of nodule formation. This project will investigate two Caesalpinioid legumes: 1) Gleditsia triacanthos, in which bacteria enter roots and fix nitrogen without nodule formation, a mutualism that probably predates the evolution of nodules, and 2) Chamaecrista fasciculata, one of the few caesalpinioid legumes that nodulates. Experiments will be carried out to determine how the bacteria establish these associations with basal legumes and identify orthologs of genes known to be critical for nodule formation in the advanced legumes. After identifying the gene orthologs, they will be mutated to test their function. A hairy root transformation system for implementing RNAi mutagenesis has been established with the help of an undergraduate student, and is a major advance for studying gene function in basal legumes. The project will provide other opportunities for student training. Caesalpinioid legumes comprise the major flora of the subtropics and tropics, serving as important food/forestry crops and replenishing nitrogen to N-limited soils. Studying these basal legumes will bring us closer not only to learning more about the genes responsible for nodulation, but also to educating students about the value of basal plants to the nitrogen economy of subtropical and tropical soils; these are threatened because of increasing human populations and climate change. Soil degradation could lead to the extinction of these environmentally important legumes.
Most people are aware of the importance of agricultural legumes, such as beans, alfalfa, and pea, but few know of how prevalent and important the basal legumes are to supplying nitrogen to soils through the process of biological nitrogen fixation. Our NSF project focuses on studying the basal legumes, which we believe will bring us much closer to understanding not only more about the broad diversity of infection and nodulation patterns in the legume family, but also help educate students and the public about the value of these plants to the nitrogen economy of subtropical and tropical soils. Our goal is to focus broadly on concepts that will lead to novel thinking about engineering plants to provide their own fertilizer. We base our analysis on investigating the species that are considered to be basal, or less advanced, in the one plant family (Fabaceae) that consistently establishes nitrogen-fixing nodules and with both alpha- and beta-rhizobia, i.e. members of the Rhizobiaceae and the Burkholderiaceae, respectively. Although the alpha- and beta-rhizobia are completely unrelated bacterial families, they interact with the plant’s genetic program in what appears to be the same manner to induce root nodules, enter the plant tissues, and fix atmospheric nitrogen into ammonia. Most of our NSF studies focused on Gleditsia triacanthos, generally considered as a non-nitrogen-fixing and non-nodulating legume. We found that nodule-like structures developed on roots and that bacteria were localized to the outside surface of these structures. We cloned a number of genes critical for both nodule formation and the mycorrhizal interaction and observed that when the plants were grown in the absence of nitrogen, they remained green, if inoculated. We also studied the infection process in Chamaecrista fasciculata, another basal legume, but one that develops bacterially infected, nitrogen-fixing nodules. Our findings show that this "basal" legume shares most of the same developmental stages as the more advanced legume species. Thus, in the basal legumes, G. triacanthos represents a transitional species in the evolution of the Rhizobium-legume symbiosis whereas C. fasciculata represents the complete conversion. By examining other basal legumes, we will learn more about how the legume family developed this ability to associate with rhizobia. Using this approach might be more productive than trying to engineer plant families that do not have the capacity to nodulate and fix nitrogen with rhizobia de novo. Our NSF-funded research is actively focused towards education and training. A postdoctoral scholar at UCLA was involved with the research as well as several undergraduate students, many of who are women or underrepresented minorities. These students represent the next generation of scientists who will make major impacts on plant biology. Although our research focused on beneficial bacteria and how the plant responds to them, some of the former Hirsch lab students are currently doing Ph.D. research on plant diseases as well as on plant-insect interactions. Another focus of study is plant breeding for improving crops not only to feed our growing human population, but also for growing plants under conditions of climate change. Dr. Hirsch is also involved in undergraduate courses that offer hands-on, discovery-based research. With student help, we have discovered several novel plant-growth promoting bacteria, which can promote plant growth either with or without rhizobial inoculation. The synergistic interactions among bacteria species living on roots or within root nodules can be considered as being identical to the synergism that exists among bacteria in the human gut microbiome. Upsetting the balance by introducing a pathogen or disturbing the normal interactions among the bacteria of either the root or gut microbiomes affects the health and well being of the whole organism.
