Plants are colonized by a diversity of microorganisms, including many that influence plant and environmental health. Plants produce a class of compounds called quaternary ammonium compounds (QACs) that are important to plant membranes and to plant tolerance to drought and salinity stress. These compounds, including betaine, carnitine, and choline, may also benefit plant-associated bacteria. For example, betaine can confer protection to stresses, choline can serve as a building block for some bacterial membrane lipids, and choline-O-sulfate may help store sulfur. Most plant-associated bacteria, including the plant pathogen Pseudomonas syringae, cannot synthesize QACs and thus are dependent on production by the host. Consequently, these organisms have likely evolved mechanisms to import and exploit these compounds. In support of this, the ability to transport choline was recently shown to provide a clear fitness benefit to P. syringae during its colonization of plants. Moreover, studies characterizing the full set of P. syringae transporters for choline and betaine uptake identified additional novel cellular components that may be involved in QAC metabolism and regulation. The goals of this project are to characterize the responses of P. syringae to specific plant-derived QACs and evaluate how these responses influence P. syringae interactions with plants. To address these goals, first, carnitine will be examined as an attractant and nutrient source by investigating the role of a newly identified carnitine-responsive chemotaxis protein in directing bacterial movement to germinating seedlings, and by identifying the genes required for carnitine catabolism and their contribution to fitness. Second, choline-O-sulfate will be evaluated as a source of sulfur and nutrients by characterizing how the loss of transport and catabolism impact P. syringae fitness on plants and by more extensively characterizing the cellular components involved in choline-O-sulfate uptake. And third, betaine, choline, phosphorylcholine and phosphatidylcholine will be evaluated as signal molecules for bacteria by examining global gene expression patterns in P. syringae mutants that lack the ability to degrade individual compounds and thus are able to accumulate them. These studies will significantly advance our understanding of how the perception, signaling and metabolism of this special class of compounds by P. syringae influences the ability of this widely studied bacterial pathogen to colonize plants. Plant-associated P. syringae populations are relevant not only as reservoirs of pathogens for plant disease, but also as reservoirs of biological ice nuclei that may have a role in atmospheric processes leading to rain. Insights into the impact of QACs on leaf-associated bacterial populations are of timely importance because increases in drought, as predicted by global climate change models, and salinity, as is occurring in large areas of irrigated agricultural lands, can result in increased QAC abundance in plant tissues, due to either natural accumulation or agricultural engineering to improve drought and salinity tolerance.
Broader Impacts This project will contribute to education by providing training for one graduate student and significant research experiences for at least five undergraduate students, several of which are expected to be members of under-represented groups in the sciences. Outreach activities will provide internships for two biology teachers (7th-12th grade) through an established program at Iowa State University for biology teachers, with the subsequent co-development and implementation of discovery-based curricula based on bacterial ice nucleation and leaf colonization. Outreach activities will also include directing at least two workshops to advance the involvement of middle school students from under-represented groups in the sciences through the Science Bound program at Iowa State University, and co-directing a workshop on plant pathogens for elementary students and their parents.
Plants are colonized by diverse microorganisms. Some of these are capable of inducing plant diseases while others provide biological control of pathogens or even promote plant growth. Plants produce a class of chemicals called quaternary ammonium compounds (QACs) that have a structural role in plant membrane lipids and a functional role in plant stress tolerance. Recent findings suggest that the plant pathogen Pseudomonas syringae lives in environments rich in the QAC choline, but prior to this project, little was known of QAC availability to plant-associated bacteria. The goals of this project were to characterize the responses of P. syringae to specific plant-derived QACs and evaluate the impact of these responses on its interactions with plants. A collection of whole-cell bacterial bioreporters for QACs was created that provide a new tool for detecting and localizing QACs in plants. These bioreporters demonstrated that choline is released from germinating seeds, seedlings and leaves of beans, making it available to bacterial colonists. The extracellular location of the activated bioreporters provided the first evidence of an extracellular choline pool in plants. This finding is significant because it indicates cell-to-cell translocation of choline, possibly during membrane turnover, and thus an opportunity for choline interception by colonizing microbes. The reduced fitness of P. syringae mutants deficient in QAC uptake demonstrated that this interception provides a significant fitness advantage to microbes that are adapted to exploit QACs. Many of the cellular components involved in QAC uptake, transformation and catabolism in P. syringae were identified, enabling the development of a library of mutants defective in various steps of QAC catabolism. Mutants deficient in carnitine catabolism demonstrated that seeds, but not leaves, release carnitine and in sufficient amounts to enhance P. syringae fitness during seed colonization. This supports a model of a seed-specific role for carnitine in plant-microbe interactions, possibly due to carnitine serving as a carrier of fatty acids during the fatty acid oxidation that accompanies seed germination. In contrast, mutants deficient in choline-O-sulfate catabolism did not show reduced fitness on seed or leaf tissues, suggesting that, at least in beans, choline-O-sulfate is not abundant and thus is unlikely to function as a sulfur source for plant-associated bacteria. This collection of mutants was used to evaluate the hypothesis that QACs function not only in catabolism and osmoprotection, but also as signal molecules for P. syringae. A global gene expression analysis of various mutants indicated that target QACs influenced only a small number of genes, many of which are involved in QAC catabolism, and therefore did not provide support for the QAC signaling hypothesis. Lastly, studies with mutants deficient in glycine betaine and phosphocholine catabolism provided insights into microbial adaptations to environments rich in available QACs by highlighting the regulation of trehalose synthesis as a particularly important point of control during extended adaptation to osmotic stress in the presence of QACs. Increasing our knowledge of the impact of QACs on leaf-associated bacterial populations are timely because drought and salinity stress, which are predicted to increase with global climate change and continued irrigation of agricultural lands, increase QAC abundance in many plant species, with further increases likely due to agricultural selection or engineering of plants to improve their drought and salinity tolerance. The results of this project indicate that such increases in QAC abundance on plants will favor the development of P. syringae populations and probably other microbes as well due to the generalized benefits of QACs. For pathogens, these populations serve as reservoirs for plant disease, whereas for beneficial bacteria, QAC-mediated increases in bacterial populations could benefit plant growth or plant health. This detailed knowledge of which QACs impact plant microbes and where those QACs are localized in plant tissues is therefore important for predicting how climate or agricultural practices affecting plant QACs will impact microbe-driven plant phenotypes. It is also important to designing strategies to manipulate plant-associated microbial populations for improved agricultural productivity. This project provided research and professional training to an assistant scientist, a postdoctoral research associate, three graduate students, six undergraduates, and two high school students, with five of these being women or members of underrepresented groups in the sciences. Through its outreach efforts, the project provided intensive research experiences for two high school biology teachers, with accompanying curriculum development, thus amplifying the number of students impacted on a continuing year-by-year basis. Through workshops and an interactive lecture, the fundamentals of plant bacteriology were shared with 13 primary school teachers, 8 secondary school teachers, 25 secondary school students, and approximately 40 primary school students and their parents.
