Sandra Clifton, Ph.D. - US grants

Plant-parasitic nematodes reduce annual US agricultural production by more than $5 billion. The most important group are the root-knot nematodes (RKN: Meloidogyne spp.), which are devastating pathogens of food and fiber crops. With the loss of chemical control agents for health and environmental reasons, the economic importance of RKN is increasing world-wide. A prerequisite to developing any new, environmentally-safe and affordable control strategy is a thorough understanding of the biology of the plant-nematode interaction. Recent advances in genomic techniques make this previously intractable host-parasite system amenable to study. Because of the intimate nature of the interaction, and the obvious developmental and physiological perturbations to the host induced by the nematode, understanding the biology of this association will shed new light on basic plant processes. The very broad host range of RKN (excess of 2,000 plant species) implies that this parasite is able to modulate some very fundamental and widely conserved aspect of host biology.

RKN hatch in the soil as developmentally-arrested larvae prior to invading a root, where the parasite establishes an intimate relationship with its host. An elaborate permanent feeding site, characterized by the formation in the host of "giant cells" within a gall, is induced by the nematode. Giant cells serve as the obligate nutritive source for the developing nematode, which becomes sedentary. Functional genomic approaches will be applied to analyze this interaction. This approach is feasible because well developed models of the host and parasite (Arabidopsis and C. elegans respectively) have been established, including complete genomic sequences.

Suites of nematode and plant genes that define pathways for establishment of the parasitic interaction will be identified, and their expression quantified. Host responses during feeding-site formation, and the biological transitions in the nematode that are coupled to the host (e.g., exit from developmental-arrest at the onset of feeding) are especially interesting. The histology suggests that phyto-hormone levels are altered in infected roots, and expression of Arabidopsis gene sets, with an emphasis on hormone-responsive genes (new members of this class are anticipated to be identified) will be quantified, as well as genes previously identified as being specifically nematode-responsive. This project addresses three general and inter-related scientific questions: 1) how is the host recognized, and how does the parasite couple its biology and development to host cues, 2) what are the parasite-induced changes in the host, and in particular what is the source and role of phyto-hormones, 3) how has parasitism evolved, what has been the role of horizontal gene transfer, and is this reflected in the organization of parasitism genes?

A large body of mostly descriptive information exists for RKN and an important goal will be to couple the genomic findings with this biological data. Many features of the RKN-plant interaction (e.g., gall formation) appear canonical for a broad range of host-parasite associations involving diverse organism (including insects), and also represents a tractable model to study aspects of normal plant development, including phyto-hormone biosynthesis/regulation. Better understanding the host-parasite interaction will reveal the linch-pins from which novel nematode-management strategies can be derived. Furthermore, because this research will be performed within a framework of graduate and undergraduate education, it will provide students with significant opportunities for professional development.

Mitochondria help to turn food into chemical energy in cells of plants and animals. Although the nucleus contains the vast majority of the cell's genes, mitochondria have their own DNA, coding for proteins vital for the survival of cells. In animals and most plants, mitochondrial genes are inherited only through the female (egg) parent. Plant mitochondrial genomes are generally much larger and more variable than those of animals. Plant mitochondria code for more proteins, including ones that can interfere with the development of pollen. This type of infertility (which does not affect female parts of flowers) is called cytoplasmic male sterility (CMS). The CMS trait can be exploited in the production of stronger and higher-yielding hybrid plants. Despite their relatively small sizes and importance, very few plant mitochondrial genomes have been sequenced.

Mitochondrial protein-coding genes appear to be very similar in different types of plants; however, most of the mitochondrial DNA lies between genes and its function and origin is unknown. Furthermore, it appears that these "intergenic" DNA sequences have little in common in different plants. Therefore, in order to understand how rapid changes occur in plant mitochondrial DNA, we need to analyze mitochondrial genomes from very closely related species. We will sequence the mitochondrial genomes from four types of cultivated maize and one of its close wild relatives, as well as from two other more distantly related monocot crop species, Tripsacum dactyloides (eastern gamma grass) and sorghum. We will identify all the potential genes and look at how they are expressed in different tissues and stages of development. The project will contribute to an understanding of the content, organization, expression and evolution of mitochondrial genomes i

0120591
Bode

It is becoming increasingly clear that understanding how an organism interacts with its environment will require a complete accounting of the genes which make up the organism and the expression patterns of the genes under various conditions. Members of the phylum Cnidaria (jellyfish, corals, sea anemones, and hydras) are key members of their environments, yet very little is known about their genetic makeup. To address this problem, a collection of cloned DNAs representing the messenger RNA populations (cDNAs) of two well-studied cnidarians will be generated. The two organisms that will be used are the freshwater cnidarian Hydra and the colonial marine cnidarian Podocoryne. Bacteria containing the cloned cDNAs will be robotically arrayed to generate archives of the clones. Sequence data will be obtained from approximately 50,000 of the cDNA clones from each organism. The resulting sequence data will be analyzed in various ways using bioinformatic computing tools. The analyses will provide information on what genes are present in cnidarians and how those genes are evolutionarily related to those in animals which diverged more recently than cnidarians, such as vertebrates and insects. Such information will be very valuable for defining the processes by which multicellular animals have evolved. The availability of cloned cDNA sets from two model cnidarians will also make it possible to examine the expression of large numbers of genes in these organisms using the technique of DNA array analysis. In particular it will be possible to identify genes whose expression changes when the organisms are placed under conditions which reflect those present during periods of environmental stress (e.g. elevated temperature). Genes whose expression levels change under stress conditions in the laboratory may be useful tools for monitoring the health of cnidarians (e.g. corals) in their natural setting.

DESCRIPTION (provided by applicant): The number of available genome sequences from the bacterial family Enterobacteriaceae is reaching a threshold where comparative genomics can drive hypotheses and experiments. In this project we have selected genomes for sequencing based on their pathogenicity and their taxonomic position. These sequences will help us understand these and other related pathogens by defining their differences and similarities in gene content. (1) The genome sequences of S. enterica serovar Paratyphi A (SPA), already sampled to 97 percent coverage, will be completed and annotated. SPA is the second most prevalent cause of typhoid and, like S. enterica serovar Typhi (STY), is restricted to humans. Typhi is undergoing genome degradation, perhaps associated with its recent adaptation to a narrow host range; we will determine if Paratyphi A is undergoing similar degradation. Klebsiella pneumoniae is a major opportunistic pathogen. We have sequenced this genome to 8-fold coverage; it will be closed, finished and annotated. (2) Cost-effective four-fold sampling (97 percent coverage) will be performed for four genomes: a biotype of S. enterica Paratyphi B (SPB), which is the third most prevalent cause of typhoid and is host-adapted to man; S. enterica Arizonae (SAR), the most distantly related S. enterica that regularly causes disease in humans; Citrobacter koseri (CKO) and Enterobacter cloacae (ECL) both of which are opportunistic pathogens representing the unsequenced genera within or adjacent to the E. coli/Salmonella/Klebsiella clade. Web-based analysis tools that take into account the incomplete nature of the samples will be used to present these data in comparison to other related genomes. Finally, (3) we have amplified and arrayed the complete open reading frames of nearly every CDS in S. enterica subspecies 1, serovar Typhimurium LT2. This resource will be supplemented with new putative CDSs, not found in STM, as these sequences become available from STY, SPA, SPB, and other serovars of S. enterica. Thus, we will develop an array that can be used in a wide variety of Salmonella, both sequenced and unsequenced, for analysis of expression and of genome content.

Obligate biotrophic plant pathogens exhibit a highly evolved relationship with their hosts, being completely dependent on living host tissue for survival. Many of the most destructive plant pathogens are obligate pathogens, including the downy mildews, powdery mildews and rusts. The maize downy mildews Peronosclerospora philippinesis and Sclerophthora rayssiae and the soybean rust Phakopsora pachyrhizi are considered major crop biosecurity threats to the US. However, genetic analysis of obligate biotrophs is difficult because they cannot be grown apart from their plant hosts. As part of an international initiative a draft genome sequence will be developed for Peronospora parasitica, a downy mildew of Arabidopsis thaliana and placed into a community annotation database. UK collaborators will carry out sequencing of ESTs and fingerprinting and sequencing of BAC clones. Pe. parasitica is a compelling choice for studying obligate biotrophy. Firstly it has culturable close relatives, such as Phytophthora infestans and Phytophthora sojae, that have excellent genome resources and are amenable to genetic manipulation, facilitating functional analysis of Pe. parasitica genes. Secondly, the host of Pe. parasitica, Arabidopsis is the leading plant model system, so that there has already been extensive physiological, genetic and genomics studies of the interaction between Pe. parasitica and its host.

Broader Impacts
The project will also create a resource for the plant-microbe interaction research community that will enable significant advances in the understanding of how biotrophic pathogens attack plants, and consequently, the design of better methods for protecting crops and natural ecosystems from these pathogens. The community annotation platform and annotation jamboree will provide multi-disciplinary training environments for postdoctoral fellows, graduate students, undergraduates from all US and international institutions.

A high-resolution, physical map of the soybean cv. Williams 82 genome is currently under construction. However, this effort is hampered by the size, complexity, and limited knowledge of the structure of the soybean genome. This Small Grant for Exploratory Research project will address the urgent need for BAC-end sequences and a public repeat sequence database to aid in map assembly and annotation. The sequence resources to be developed will provide a better estimate of the gene and repeat content of the soybean genome. The BAC-end sequences will be integrated into the physical map, as well as used to anchor genes and genetic markers.

The specific goals of the project are to 1] generate a repeat sequence database by single-pass sequencing of 25,000 randomly generated plasmid clones, 2] sequence 20,000 BAC-ends, 3] annotate the sequence, and 4] incorporate the data into the Fingerprint Contig (FPC) database for community access (http://www.genome.arizona.edu/). Sequence data will be released to the GSS division of GenBank (http://www.ncbi.nlm.nih.gov/dbGSS/) on a weekly basis.

A robust map of the soybean genome will facilitate the development of a wide range of functional genomics tools for soybean. An additional outcome will be the recruitment of young scientists who will see soybean as an exciting research system on which they can build their careers. This increase in critical mass will have many tangible benefits, including preserving the viability of soybean as an agriculture crop and contributing to homeland security by insuring a safe and available food supply.

The widespread view of bacteria as primitive asocial organisms has been drastically altered in the past decade by the finding that, using a process called quorum sensing, bacteria communicate using mixtures of secreted chemical signals. Quorum sensing allows bacteria to synchronize gene expression, and therefore behavior, on a population-wide scale, and thus allows them to take on characteristics once assumed to be restricted to higher organisms. Vibrio harveyi, a free-living bioluminescent marine bacterium, has been at the forefront of this paradigm shift, because it was the first bacterium shown to communicate with multiple chemical signals and to be able to communicate across species boundaries. This research will determine the complete V. harveyi genome sequence. The genome sequence will enable an understanding of intra- and inter-species cell-cell communication and how bacteria integrate, process, preserve, and react to multiple sensory inputs. Comparing the V. harveyi genome sequence to other sequenced Vibrio species (Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus and Vibrio fischeri) should enhance understanding of the unique biology, ecology, and disease determinants of this clinically and economically important group of aquatic pathogens. The broader impacts of this work are that quorum sensing studies are providing insight into information processing at the cellular level, the evolution of multi-cellular organisms, and population-level cooperation. The PI teaches undergraduate and graduate courses and she is the Director of Graduate Studies. She sits on many committees and panels, performs editorial duties, runs several science outreach programs, and is an active research mentor.

[unreadable] DESCRIPTION (provided by applicant): S. enterica subspecies I contains over 1500 different serovars with an extraordinary diversity of host ranges and pathogenic mechanisms. This continuing project will generate a resource of sequenced genomes and corresponding phenotypic data of judiciously chosen S. enterica serovars to capture some of this diversity. In the previous funding period, reductions in sequencing costs allowed us to completely sequence three Enterobacterial genomes instead of the two originally proposed. A recent leap in sequencing technology will allow the near complete sequencing of an additional 25 S. enterica genomes at the same cost. Strains will be picked for both their clinical and their taxonomic importance. Previous project results taught us that Salmonella subspecies I serovars typically share a backbone of about 90% of their -4500 chromosomal genes. The remaining 10% of each genome in these 25 strains may contain an additional 4000 new or considerably divergent genes, including prophage, some of which contain multiple embedded virulence genes. The genome sequences will permit unprecedented resolution in the study of accelerated evolution of surface markers, such as lipolysaccharide and fimbrial genes, and lateral gene transfer events. The project will also acquire data on host-range, virulence, and in vitro phenotypes for 96 strains, including each sequenced strain, to identify laboratory models to study these strains. Sequence, annotation, and phenotypic data from a large number of S. enterica strains will be made publicly available and will lay the groundwork to allow the study of mechanisms responsible for host range, pathogenic mechanism, the emergence of new variants, and will provide information that can be exploited for novel therapy development. [unreadable] [unreadable] [unreadable]

Fungus-growing ants and their microbial partners form a complex symbiosis, involving at least four integrated, coevolved, and anciently associated lineages that span three taxonomic kingdoms. At the center of the symbiosis is the obligate mutualism between the ant farmers and the fungi they cultivate for food. The ant-fungus mutualism is parasitized by microfungi in the genus Escovopsis, and to defend against infection, the ants have a mutualistic association with antibiotic-producing filamentous bacteria in the genus Pseudonocardia. The antibiotics produced by Pseudonocardia suppress the growth of the specialized parasite Escovopsis, and these compounds differ depending on the strain of bacteria. The genomes for three strains of Pseudonocardia that span the diversity of the mutualism will be sequenced, as well as the genome of P. saturnea, a closely related non-symbiotic species. In addition, optical maps for the genomes of 20 Pseudonocardia strains will be generated, which will facilitate estimations of the degree of genome plasticity and rates and patterns of evolution in this group of bacteria. Symbiosis is a major theme in the history of life; thus, this work will contribute to understanding general mechanisms that drive biological complexity and diversity. The research team will contribute to education and training through the development of public displays related to this project for The Microbe Place in the Microbial Sciences Building at the University of Wisconsin-Madison, as well as through a yearly workshop on microbial genomics for undergraduate and graduate researchers.