Joy Bergelson, PhD - US grants

There have been several recent theoretical and empirical studies of competition between annual plants. Despite these efforts, many empiricists and theoreticians agree that there is little understanding of the impact of spatial heterogeneity on competitive interactions between plants. This is disturbing given the patchy nature of plant distributions and the ability of patchiness and dispersal to markedly alter competitive relationships in simple discrete models. The following are lacking: 1) experiments in which the spatial distrubutions of inter- and intraspecific competitors are manipulated; 2) experiments in which the consequences of dispersal (which alters these distributions) are investigated; and, 3) a theoretical framework for investigating competitive interactions between species with nonrandom spatial patterns. This project will investigate how spatial distributions and seed dispersal influence the dynamics of competing populations of Poa annua and Senecia vulgaris. A series of single generation competition experiments in which spatial distribution is manipulated under controlled conditions will be performed. Also the effects of dispersal will be incorporated by performing field experiments in which spatial distributions and dispersal are minipulated over many generations. Parallel with these experiments a model to explore more general questions about how dispersal and spatial patterns interact to determine plant population dynamics.

9623387 Bergelson Plants are known to produce a wide variety of chemical compounds, some of which are used in medicine today. Many theories explaining the diversity of these compounds propose that plants produce them as defenses against herbivorous insects, but there is little experimental work to demonstrate the validity of this idea. This award will fund experiments to address the role of a particular class of plant chemicals, the tropane alkaloids, produced by members of the potato family (Solanaceae). Three strategies will be adopted to determine the functional importance of the major tropane alkaloids using two closely related members of the Solanaceae, deadly nightshade and jimson weed. The first approach is to add the alkaloids, both individually and in mixtures, to insect artificial diet in order to ascertain their effects on insect growth and survival. The second strategy is to test whether combinations or amounts of the alkaloids showing greatest toxicity to insect herbivores in the laboratory have a defensive function in natural populations. The final strategy is the most novel, and takes advantage of recent biotechnology to insert genes into plants. A gene for one of the enzymes necessary to create the alkaloids will be inserted into plants in the laboratory, qualitatively changing their alkaloid production. Individuals having these 'mutant' alkaloid profiles will be planted out in the field with ordinary or wild type plants, and the amount of damage and total seed production for plants of both kinds will be measured. This project has both implications for our understanding of the evolution of plant chemistry, and for the applied management of crmps. In particular, findings will help guide genetic engineering of more resistant plants and, in so doing, allow farmers to be less dependent on pesticide application.

Kreitman 9800957 Plant species generally exhibit variation for resistance to disease. This striking observation implies that a balance of opposing forces acts to maintain variation for disease resistance. Since the cost imposed by disease would drive populations toward increased resistance, a trade-off should oppose increased resistance to account for the observed variation. In the model plant species Arabidopsis thaliana, a balance of opposing forces maintains molecular variation at disease resistance gene Rpm1. The proposed research will investigate the origin and determinants of variation for disease resistance within populations. High through-put methods will allow genotyping of hundreds of A. thaliana individuals from local populations in Indiana and Michigan. Analyses will measure associations between Rpm1 and nearby genetic markers. Little association would support a balance within populations, while greater association would support a balance due to migration among populations where different forces dominate. Arabidopsis disease resistance has come under intense study recently, and the ecological and evolutionary work proposed here will complement molecular genetic work on this system. Plant disease resistance must be understood to facilitate the management of disease in crops, for assessing planting strategies and predicting the impact of disease in natural plant populations and weeds. Furthermore, plant disease resistance can be compared with the animal immunity for a more general understanding of the pressures pathogens place on host species. This project continues in the vein of Kreitman's work on the fruit fly Drosophila, at the interface between population genetic theory and empirical data. Arabidopsis Rpm1 is a strong example of balancing selection maintaining molecular variation. Continuing study of Rpm1 will help elucidate the patterns and processes shaping genetic variation and change. Finally, the proposed work continues to develop Arabidopsis as an important new model organism for population genetics. Arguably, no other model organism affords as much potential for interplay with ecology and genetics, both well-established fields in Arabidopsis biology.

In A. thaliana, alleles of R loci confer resistance by interacting with specific avirulence (avr) gene-dependent products of bacterial or fungal pathogens. The rapid progress being made in the molecular and functional characterization of R genes provides a timely opportunity to investigate the ecological or molecular population genetics of resistance in this species: essentially nothing is known about the extent of polymorphism at R gene loci, the age of alleles, or the geographical distribution of polymorphism. We propose to investigate why many R gene loci are polymorphic for two or more functional alleles by utilizing two complementary approaches for studying selection acting on resistance loci. First, we will perform a statistical analysis of DNA sequence polymorphism within and among populations (and species) at five R loci and along two chromosomes. Second, we will experimentally estimate the relative fitness of resistant and susceptible alleles at two loci, Rpm1 and Rps5, by constructing transgenic and control lines of A thaliana. Together, these studies will allow us to test specific selection models for the maintenance of polymorphism and the evolution of R genes in A. thaliana, and will allow us to make inferences about the spatio-temporal population dynamics of these host-pathogen interactions. Our data will enable us to test the widespread belief that there will be a rapid selective turnover of R alleles resulting from an evolutionary arms race between pathogen and host. We will also test for evidence of long-term evolutionary maintenance of allelic variation at R loci, a prediction made by a subclass of theoretical models of gene-for-gene coevolution. These tests require information on background levels of polymorphism, which we will generate through a systematic survey of nucleotide polymorphisms along two chromosomes of A. thaliana. Information on the extent and spatial patterning of molecular genetic variation is integral to testing for historical evidence of selection at R loci, and will help establish Arabidopsis as a model system for molecular evolutionary studies on a wealth of plant traits.

Bergelson
9972708

R-genes in plants determine whether individuals are resistant or susceptible to particular strains of disease causing pathogens. R-genes tend to be quite variable in natural plant populations, yet the forces maintaining this genetic variation are not well understood. One hypothesis is that susceptible plants produce more seed than resistant plants under certain environmental conditions. In this way, resistance is not always favored and both phenotypes are maintained. The experiments detailed in this research test the hypothesis that the energetic cost of having a resistance response to a pathogen is greater than the benefit the plant receives from stopping pathogen growth under some circumstances. If R-gene resistance responses are costly, then the level of resistance response may determine whether a resistance response has a net benefit or cost.

In addition, this project will investigate whether different levels of resistance response have different benefits and costs for the plants. Arabidopsis thaliana infected in the greenhouse with its natural pathogen, downy mildew, will be investigated. Plants that differ by the presence/absence of an R-gene but are otherwise genetically identical will be compared. In combination with theoretical investigations, these experiments will explore whether the level of R-gene mediated resistance is an important factor in the coevolutionary interactions between A.thaliana and downy mildew.

These experiments will provide fundamental knowledge about plant-pathogen coevolutionary interactions. Knowledge about these interactions in natural populations will improve the application of pathogen resistance in agricultural plant species.

0107219
Bergelson

Plants can recognize gene products produced by pathogens, and this recognition triggers a strong defensive response that severely limits the pathogen's growth in the plant. An important question in the area of plant disease, is "Why do pathogens carry genes which ultimately trigger a defense response against them"? In this Doctoral Dissertation Improvement Grant, the researchers propose to look for benefits associated with the pathogen genes that elicit plant defenses. They propose to take a plant pathogenic bacteria and remove several of the elicitor genes, and then compare the performance of the original strains (which have all the genes) to the mutant strain (which possesses a subset of the genes). The researchers will investigate several factors including disease pathology, epidemiology, and survival, under both greenhouse and natural field conditions.

Agriculture and the plant biotechnology industry are very interested in pathogen defense elicitor genes. It is now possible to engineer plants to express new resistance genes that recognize the elicitor genes of common plant pathogens. Before such a strategy is attempted, it would be valuable to understand how the elicitor genes function and are maintained in pathogen populations. If elicitor genes can be removed with little or no fitness cost to the pathogens, then a biotechnology strategy that depends on the expression of elicitor genes can easily fail.

This proposal links the molecular biology of disease genes to the population-level processes that determine the frequencies of these genes and their evolutionary history. The host organism is the plant Arabidopsis thaliana and the pathogen is Pseudomonas viridiflava. The proposal focuses on host loci that affect resistance or susceptibility and pathogen loci that affect virulence (successful infection) or avirulence (unsuccessful infection). The first aim begins with measuring the phenotypic patterns of infection success or resistance for different host plants and pathogen isolates. In each of 10 populations, the investigators will analyze the outcome of infection for 50 pathogen isolates tested against each of 10 host genotypes. Next, three pathogen loci that affect virulence will be cloned. The three loci will be chosen based on their different infection successes when tested against host genotypes that have been well characterized at the sequence level and that provide helpful molecular tools for later analysis. Once the three pathogen loci have been chosen, the investigators will search for three matching loci in the host plant that interact with the pathogen virulence loci. Matching gene-for-gene interactions between plant and pathogen have been frequently observed and are likely to be found in this case. The final step for the first aim measures epidemiological aspects of natural populations. In particular, the investigators will study various natural populations for infection rates, host population densities and migration rates, and patterns of DNA polymorphism in the three host and pathogen loci that have been cloned. The second aim develops mathematical models. These models will be used to formulate hypotheses about how the population biology influences the frequencies of host and pathogen alleles and the pattern of molecular evolution at the cloned loci. The models emphasize how epidemiological processes of infection frequency and spread of disease influence aspects of gene frequency and molecular evolution. The third aim will use the data generated to estimate parameters of the mathematical models. Estimates include rates of epidemiological spread of disease, the fitness differences between plants that have or lack particular resistance alleles (cost of resistance), the fitness differences between pathogens that have or lack alleles that allow them to attack particular plant genotypes (costs of virulence), migration rates between populations, and the effects of environment (e.g., humidity) and host density on rates of pathogen transmission. The fourth aim uses the parameter estimates to expand the mathematical models and to study various aspects of epidemiology and evolution. For example, if weather has a significant impact on transmission, then that factor will be incorporated into the epidemiological components of the model. The model will be tested in the sense that the investigators will search for consistent explanations for how observed patterns of polymorphism, molecular evolution, and epidemiology fit together. For example, the epidemiology along with costs of resistance and virulence allow estimates for tendency of allele frequencies to fluctuate over time. The tendency of allele frequencies to fluctuate has, in turn, consequences for the expected patterns of molecular evolution. Thus, the investigators can use a particular aspect of their data to estimate processes such as the tendency of allele frequencies to fluctuate. They can then use their estimate of allele frequency fluctuations to make testable predictions about observable patterns, such as the distribution of nucleotide polymorphisms.

Abstract
DISSERTATION RESEARCH: Evolutionary Dynamics of a Natural Plant-bacterial Pathogen Interaction
Bergelson
DEB-0309028

Ecological processes and genetic variation are increasingly being integrated in coevolutionary models of plant-pathogen interactions, yet little progress has been made in relating this theoretical work to natural systems. This research will characterize the dynamics of the interaction between the bacterial pathogen Pseudomonas viridiflava and the plant Arabidopsis thaliana by combining experimental and observational data with an evolutionary model. This will be one of only a few studies of natural plant-pathogen interactions and the first to study the dynamics of a bacterial pathogen in a wild plant population. A. thaliana has been a highly productive model system for studying plant resistance to pathogen infection in the laboratory and a growing amount of molecular evolutionary data is available for genes in A.thaliana that confer resistance to bacterial pathogens. By placing disease resistance in A. thaliana in an ecological and evolutionary context, this study will contribute to a greater understanding of recent molecular genetic results. A more complete understanding of the evolutionary dynamics of resistance in plant populations and virulence in pathogen populations, through studies such as this one, will be a necessary step towards the achievement of durable disease resistance in agricultural crops.

The main objective of this project is to develop tools and resources that will enable the community of Arabidopsis geneticists to carry out population surveys for marker-trait associations - linkage disequilibrium mapping. The basic idea is simple: rather than mapping genes by studying crosses, one types a large number of unrelated individuals (1,152 in the present case) with respect to a large number of variable marker loci (6,144 in the present case) distributed across the genome, in order to identify chromosomal regions that appear to be shared by individuals that are phenotypically similar (in the sense of being resistant to a particular pathogen, for example). This approach potentially leads to much faster gene identification than traditional methods, but has only become practicable as a result of advances in technology for studying genetic variation.

The project is part of the effort to understand the genetic basis for phenotypic variation - arguably the greatest challenge facing modern biology, and central to genetic epidemiology (e.g., why some people are more susceptible to asthma), plant and animal breeding (e.g., why some strains of rice more tolerant to drought), as well as basic evolutionary biology (e.g., the kinds of genetic changes underlie adaptation to a novel habitat). While the project is directed toward the model plant Arabidopsis thaliana, the methods that will be developed are broadly applicable (including to humans). Furthermore, Arabidopsis is a model for plant biology, and is often used to study agriculturally important traits indirectly.

The environment in which organisms live is not static but changes as a consequence of human activities, as well as geological and evolutionary processes. One particularly dynamic environment is that of a biological host within which microbes live. In these environments, change can be rapidly induced by the presence of the microbes themselves when they trigger host resistance responses. How does this environmental change shape the ecology and evolution of microorganisms, and how do they survive in such fluctuating environments? The goal of this project is to dissect the ecological and evolutionary responses of bacteria that live in the leaves of a host plant, A. thaliana, to changes that are induced in host plant chemistry. In so doing, this project will attempt to (1) identify the community of bacterial species that reside in this important host plant, (2) explore the community level interactions of these species as a function of biochemical changes in the host, particularly making use of genetic mutants that differ in their capacity for defense responses, (3) examine genome-wide patterns of genetic variation in an attempt to infer the evolutionary history of these species and (4) experimentally test the evolutionary capacity of microbes to adapt to novel host environments. The project is thus integrative, encompassing ecology, evolution, genetics, genomics and biochemistry, in an effort to develop a mechanistic understanding of biological response to a changing environment.

Most, if not all, species are faced with changing environments, although the timescale of this change varies. Of particular practical concern is whether we can predict the capacity of species to adapt to novel environments, and whether we can anticipate the extent of community level compositional change. Discoveries about the role of plant defense on the resident microbial community in ecological and evolutionary time should yield general principles that will be applicable across a broad spectrum of microbial communities. This project aims to discern these general principles. The project will also serve important educational missions, including (1) a role for high school and undergraduate students in the research, (2) a component to train K-12 teachers in microbial ecology, (3) efforts to include both a technician and graduate students from an under-represented group, and (4) hands-on presentations on biodiversity to nursery and elementary school students, including display of student made installments at The Field Museum in Chicago.

Local adaptation is characterized by natural selection for traits that improve fitness under local conditions, irrespective of consequences of these traits in other habitats. Scientists have studied local adaptation via reciprocal transplant experiments: planting individuals of the same species from two different locations together, then assessing if individuals grown at their home site show higher survival and reproduction. However, the extent to which underlying genetic changes contribute to local adaptation remains poorly understood. This research aims to illuminate the genomic basis of local adaptation by pairing reciprocal transplant experiments with genome-wide genetic analysis, using Arabidopsis thaliana plants from different habitats in Sweden.

The novelty of this study lies in two areas: the application of cutting-edge genomic resources to plant populations growing in native habitats, and the ability to pinpoint specific regions of the genome associated with survival and reproduction in the field. The researchers will disseminate results, information on rare and isolated populations of A. thaliana, and seed stocks widely to the scientific community. Finally, the value of this research becomes clear when considering the challenges of global climate change, conservation, and habitat restoration in the modern world, all of which touch on the issue of adaptation to specific environmental conditions.

DESCRIPTION (provided by applicant): One of the most important challenges facing biology today is making sense of genetic variation. Understanding how genotypic variation translates into phenotypic variation and how it is structured in populations is fundamental to our understanding of evolution, and has enormous practical implications for human health as well as for agriculture and conservation. The long-term objective of this project is to increase our understanding of the molecular genetic basis for adaptive variation by studying flowering time in A. thaliana. The focus is on the flowering response to cold temperatures, so-called vernalization, which is one of the major mechanisms plants use to ensure that they flower at the right time, during the right season. The project seeks to describe the genetic architecture underlying variation for this trait, and will identify, at the molecular level, the major genes and alleles involved. The adaptive significance of the identified polymorphisms will be determined in field trials. The project has three specific aims: First, to map the genes responsible for variation between plants collected in different parts of the world. The mapping will be done using a combination of traditional linkage mapping methods and so-called genome-wide association scans, in which modern genotyping technology is used to survey massive amounts polymorphisms in population samples in order to identify genomic regions that appear to be statistically associated with the trait. Second, molecular genetics will be used to characterize the identified alleles and loci. Third, the pattern of variation in and around identified loci will be analyzed in order to elucidate the history of selection on the loci. Project Narrative: One of the most important challenges facing biology today is understanding how genetic variation between individuals translates into variation we can see or measure, like blood pressure in humans, or drought tolerance in rice. The goal of this project is to increase our understanding of the general principles that underlie the genetics of adaptive natural variation by studying flowering time in the model plant thale cress (Arabidopsis thaliana). The focus is on the flowering response to cold temperatures, so-called vernalization, which is one of the major mechanisms plants use to ensure that they flower at the right time, during the right season.

The hybridization of genetically distinct groups is an important, natural occurrence in many plants that can generate new species and transfer beneficial traits from one group to another. Genetic information, such as which and how many genes affect hybridization, is lacking for most plants, including many ecologically and economically important species. This research will use state-of-the-art DNA sequencing methods to examine the natural hybridization of two ecotypes (genetically distinct varieties adapted to different habitats) of switchgrass, a perennial tallgrass native to North America. These DNA sequencing methods generate hundreds of times more data than previously possible, thus enabling analyses across the entire genome of switchgrass rather than just a handful of genes. The data will be used to identify hybrid populations, analyze patterns of migration, and identify regions in the switchgrass genome that are being transferred between ecotypes or that decrease the success of hybrids.
Switchgrass is an emerging bioenergy crop and one of the most widely used species for land conservation. The patterns of genetic variation and hybridization identified in this project will have direct implications on selecting appropriate varieties of switchgrass for bioenergy production and conservation needs. Additionally, the characterized hybrid populations will be valuable resources for future work identifying genes that affect economically important bioenergy traits. Beyond the bioenergy and conservation implications, this proposal will act as a template for implementing new genotyping methods in other ecologically and/or economically important species that, like switchgrass, do not have the research resources of traditional model organisms.

Bacteria colonize diverse environments, and the various traits that allow them to survive in these different environments are of wide ecological, medical and agricultural relevance. There is thus considerable interest in determining the genetic changes that allow specific strains to flourish in some environments while perishing in others. This project will identify the genes that allow the plant bacterial pathogen Pseudomonas syringae to spread in different environments. The proposed study pairs the comparison of bacterial genomes with comparative functional genetics to identify the specific genes that render P. syringae strains acute pathogens in crop populations and natural plant populations and allow P. syringae to proliferate in soil.
An understanding of the genetic changes that allow bacteria to survive in different environments has important implications because slight genetic changes in a bacterial strain can result in the decimation of entire plant populations or the death of a patient. The cross-disciplinary nature of the project supports the training of undergraduate and graduate students. The project includes outreach and education of K-12 students, including students from underrepresented groups.