Brett M. Tyler - US grants

Gene regulation is fundamental to the problems of cell differentiation and tissue morphogenesis, including cancer. Neurospora crassa possesses a well-developed genetic system, including DNA transformation. These have enabled extensive genetic analysis of several regulated N. crassa genes, providing detailed models of transcriptional regulation for more complex eukaryotic systems. Recently I developed an RNA polymerase II in vitro transcription system for N. crassa, enabling biochemical analysis to be extended to fungal transcriptional regulation for the first time. The aim of this project is to identify and functionally characterise the basic protein and DNA components of the N. crassa RNA polymerase II transcription apparatus, in order to provide a framework for defining biochemically how genetically characterized fungal regulatory proteins modulate transcription. Specifically, the aims of the project will be: (i) Using the strongly expressed 'housekeeping' gene, am (glutamate dehydrogenase) as a model, N. crassa RNA polymerase II transcription factors will be isolated from the existing crude in vitro system. The factors will be identified and then assayed during isolation by their binding to specific DNA sequences and/or by their requirement in a transcription assay containing separated protein fractions. (ii) The promoter DNA sequences required for am transcription will be determined. These sequences will be identified by their interaction with specific transcription factors, and by creating systematic sets of promoter mutations whose effects will be tested using in vivo and in vitro transcription assays. (iii) The different steps in the formation of an N. crassa RNA polymerase II transcription initiation complex, and the factors required for each step, will be determined by order-of-addition experiments, and by using inhibitors of specific steps in transcription initiation. (iv) The possibility of developing in vitro transcription systems for genes from other fungi such as S. cerevisiae, S. pombe and A. nidulans will be explored using N. crassa transcription factors either alone or in combination with proteins from these other fungi.

The overall aim of this project is to identify mechanisms by which the transcription of ribosomal RNA and protein genes is co- ordinated in eukaryotes, using Neurospora crassa as a model system. Previously this lab has shown that Neurospora crassa 5S and 40SrRNA genes require a common sequence, the ribo box, for transcription in vitro, and that a Neurospora ribosomal protein gene crp-2 contains three ribo boxes in its 5' flanking sequences. This proposal is designed to test the hypothesis that the protein that binds to the ribo box co-ordinates the transcription of 5S rRNA, 40S rRNA and ribosomal protein genes in Neurospora. It is proposed to test the regulatory role of the ribo box by placing rRNA and r-protein genes with ribo box mutations back into Neurospora and measuring their transcription and regulation in vivo, and by introducing ribo boxes into unrelated genes such as qa-2 and tRNAleu, and testing the effects of those additions on the regulation of those genes in vivo. To examine the molecular action of the ribo box it is proposed to purify the ribo box-binding protein and to characterize its properties, and to attempt the cloning of the gene coding for this protein.

EIA-0131899
Dickerson, Allan
Virginia Polytechnic Institute and State University


SUMMARY

Cells routinely perform complex computational tasks that enable them to control the orchestration
of thousands of genes and communicate with other cells to manifest emergent properties such as growth
and differentiation.Understanding and engineering the algorithms underlying the complex computational
machinery of cells should have significant impact in science and technology,particularly biotechnology,
biocomputation and medicine.

Several notable recent reports demonstrate that it is possible to design and construct simple de
novo genetic circuits such as a switch and an oscillator in Escherichia coli .This work also revealed that
implementation of even the most simple circuits in vivo requires tedious optimization of often poorly-
understood protein-DNA interactions and mRNA and protein stabilities,among other parameters.We
propose to develop efficient,evolutionary design strategies for constructing functional de novo genetic
circuits.We will apply methods of molecular evolution,which have proven highly successful for
engineering proteins with improved or altered properties,to complex genetic systems involving multiple
repressors,operators,and promoters.We believe that evolution will prove to be generally applicable for
optimizing individual devices as well as complex genetic circuits,and our goal will be to demonstrate
how evolutionary searches are best performed in order to build libraries of devices and assemble them
into functional circuits.

Because of their economic impact as plant pathogens, Phytophthora species are being intensively studied through molecular genetic and genomics approaches. These organisms have served as lead species for the entire Stramenopiles lineage, a major radiation of crown eukaryotes distinct from plants, animals and fungi. The Phytophthora molecular genetics community has developed a strong culture of collaboration, communication, and sharing techniques and resources. With the recent blossoming of genetic and genomic tools for Phytophthora, many new investigators from a variety of backgrounds have become interested in Phytophthora molecular genetics. This award will facilitate the integration of these investigators into the community and will further strengthen the cooperative culture of this community. The specific goals of the network follow:
1. To promote communication and collaboration, and to minimize duplication of effort, within the worldwide Phytophthora molecular genetics community by means of an annual workshop, exchange visits among laboratories, and a community web site.
2. To produce and distribute research resources including libraries, specific DNA clones, genetic strains and cDNA microarrays, that will benefit the whole community,
3. To promote the entry, participation and training of students, postdoctoral fellows, junior faculty, and more established faculty in the field of Phytophthora molecular genetics. The network is especially interested in facilitating the participation of minority individuals and those from predominantly undergraduate institutions, institutions with high percentages of students from groups underrepresented in the U.S. research infrastructure, and institutions in states that have traditionally received lower amounts of federal funding for research and development. This will be done by means of travel awards to the annual workshop, training internships, and facilitated access to techniques and resources. Faculty from nations without established research infracstructures will also be eligible for some support.

This Research Coordination Network is open to all researchers with an interest in Phytophthora molecular genetics. It promises to broaden the diversity of scientists working in this field and to advance our understanding of the biology of these important plant pathogens.

Some plant disease resistance genes (major resistance genes) confer high levels of resistance against particular pathogens. These genes have been well studied over the past decade. Major resistance genes, however, are generally effective only against specific strains of a pathogen, and so become ineffective in the field when new pathogens strains appear. In contrast, partial or quantitative resistance is effective against all strains of a pathogen, albeit providing a lower level of protection. Multiple genes confer partial resistance, each making minor contributions, complicating the study of these genes. As a result, less is known about how they operate.

The goal of this project is to understand the mechanisms of quantitative resistance of soybean against the oomycete pathogen Phytophthora sojae, which is one of the most damaging soybean diseases. Oomycetes are fungus-like organisms that are actually most closely related to brown algae such as kelp and diatoms. Phytophthora pathogens attack thousands of plant species, including many important crops.

Two approaches will be combined to identify and characterize quantitative resistance genes. First, genetic crosses between soybean cultivars differing in their quantitative resistance to Phytophthora will be analyzed to identify genetic loci (called quantitative trait loci or QTL) in soybean that contribute to resistance. Second, the expression levels of thousands of soybean and Phytophthora genes during infection will be assayed using hybridization microarrays to determine the mechanisms of resistance contributed by different quantitative resistance loci. Pathogen gene expression patterns will be analyzed to determine the impact of plant defense mechanisms on the pathogen. Overall, it is anticipated that this research will result in new insights into plant defense mechanisms, new genetic and genomic resources for soybean researchers and breeders, and new statistical tools for analysis of gene expression data.

Drs. Brett Tyler and Bruno Sobral of Virginia Tech., in collaboration with Dr Jeff Boore of the DOE Joint Genome Institute have been awarded a grant to create a rough draft of the entire DNA sequence of the soybean pathogen Phytophthora sojae. Plant pathogens from the genus Phytophthora cause destructive diseases of an enormous variety of crop plant species as well as forests and native ecosystems. The potato pathogen P. infestans was responsible for the Irish potato famine, and is still a destructive pathogen of concern for biosecurity. Superficially, Phytophthora pathogens resemble fungi, but they in fact belong to a group called Stramenopiles that are most closely related to algae such as kelp and diatoms. Hence conventional fungus control measures often fail against these pathogens. P. sojae has been chosen for the project because it is relatively easy to genetically manipulate. This project will be tightly integrated with a project funded by the DOE to determine a draft sequence of the oak sudden death pathogen P. ramorum.

The broader impact of the project will include direct training of postdoctoral fellows and undergraduates, and visiting researchers from minority institutions in a multidisciplinary, team-oriented environment that emphasizes development of the communications skills required for synergistic interactions between experimental and computational biology. The community annotation platform will provide a multi-disciplinary training environment for researchers world-wide. This research will advance the microbial genome research infrastructure by developing and making publicly available, the first in-depth genomic resource for a pathogen from the Stramenopile kingdom. This resource will provide a basis for new control measures against a group of pathogens which have major impacts on global ecosystems, food security and agricultural production.

This is a Microbial Genome Sequencing Award funded through a collaborative program between the National Science Foundation and the Department of Agriculture.

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 wide diversity of microbes live on or in the tissues of plants, including bacteria, fungi, nematodes and fungus-like organisms called oomycetes. All of these microbes, whether beneficial or pathogenic (disease-causing), have developed mechanisms to neutralize the defense systems used by plants to protect themselves against infection. Understanding the similarities in the ways these diverse microbes attack plants will help us understand the vulnerabilities of plants to infection, which will in turn help design new and effective methods for controlling plant disease. Currently, genome projects focused on a variety of plant pathogenic microbes are uncovering large numbers of genes that the microbes may use to attack plants. However, because these genome projects are going on in different research communities, the terminology that is used to describe the functions of the genes varies so much that it is very difficult to compare the functions of genes in the different microbes. This project will establish a standardized set of terms, called a Gene Ontology, for describing the functions of genes used by microbes that live in or on plants, including pathogenic microbes. The work of establishing the terms will be carried out in collaboration with the Gene Ontology Consortium, which is a group of institutions involved in creating standardized terms for describing all the functions of genes in living organisms.

The broader impact of this work will be to establish a means for greatly facilitating the exchange of information among researchers studying plant-associated microbes. The work will also be of value to researchers studying animal- and human-microbe interactions. Members of many different research communities will be trained in the use of the standardized terms, through training workshops, internships, presentations at meetings, and on-line training documents. Training will emphasize graduate students, postdoctoral fellows and junior faculty, especially those who are minorities, or are from undergraduate institutions. Information about the standardized terms will also be incorporated into courses for graduate and undergraduate students.

This project is being supported through a collaboration of the National Science Foundation and the National Research Initiative of the Cooperative Research, Education, and Extension Service of the Department of Agriculture.

Oömycetes, or water molds, comprise a diverse group of heterotrophic organisms that morphologically and physiologically resemble fungi. Many oömycetes are saprophytic, especially in aquatic habitats. More commonly though, oömycetes are destructive pathogens of plants and animals. The last five years have seen a rapid expansion in resources for oömycete genomics and a rapid growth in the number of researchers interested in oomycetes. This project will strengthen the strong culture of collaboration and communication within that community and facilitate the sharing of techniques and resources.


The central thrust of the program is to ensure that the transformation of the oömycete molecular genetics community into a genomics, bioinformatics, and molecular genetics community carries all the community research groups with it, including less well-funded groups that are disproportionately located at small minority-serving or primarily undergraduate-oriented institutions, or at institutions in nations with poorly developed research-funding infrastructures. Furthermore, the entry, participation and training of new investigators into the field of oömycete genomics, particularly junior faculty and faculty from institutions under-represented in the U.S. research infrastructure, will be promoted. The mechanisms to promote these goals include an annual workshop, training workshops, brief training internships focused on bioinformatics, a web site, and a resource center to maintain and distribute materials for the worldwide oömycete genomics community.

Oomycete plant pathogens, such as those from the genus Phytophthora, cause destructive diseases of an enormous variety of crop and non-crop plant species. Forests and native ecosystems have been devastated by broad host range Phytophthora species such as Ph.cinnamomi and the newly emerged pathogen of California's oak forests, P. ramorum. The potato pathogen Ph. infestans was responsible for the Irish potato famine in the 19th century. Superficially, Phytophthora pathogens resemble fungi, but they in fact belong to a group called oomycetes that are most closely related to algae such as kelp and diatoms. Hence conventional fungal control measures often fail against these pathogens. Since these organisms have evolved the ability to attack plants independently of other pathogens such as fungi, they may have developed novel means of attacking their hosts. This project, which will also be funded by the USDA/CSREES, will create a comprehensive genetic resource for oomycete pathogens by creating a high quality DNA sequence of the soybean pathogen Phytophthora sojae. The Ph. sojae sequence will be integrated with a draft sequences for five other oomycete pathogens, Ph. ramorum, Ph.infestans, the pepper and cucurbit pathogen Ph. capsici, the downy mildew Hyaloperonospora parasitica, and the broad host range opportunistic pathogen Pythium ultimum.

The project will greatly advance understanding of the genetics, biochemistry, evolution and pathology of this destructive group of pathogens and will facilitate the development of improved disease control measures. A network of predominantly undergraduate institutions will be involved in interpreting the genome sequences, providing exciting opportunities for undergraduates to experience the excitement of exploring the cutting edges of biology.

Oomycete plant pathogens cause extremely destructive diseases on crops and in natural ecosystems. Little is known about the molecular weapons that enable oomycetes to be such successful pathogens. Analyses of four recently completed oomycete genomes suggest that oomycetes export dozens or hundreds of their own 'effector' proteins to the interior of plant cells. These pathogen effectors are expected to sabotage plant immune responses and trigger other effects that benefit the pathogen. However, almost nothing is known about the molecular functions of oomycete effectors. This project will investigate the functions of six pairs of effectors that are conserved between two distantly related oomycetes: the Arabidopsis downy mildew pathogen Hyaloperonospora parasitica and the soybean root rot pathogen Phytophthora sojae. It is expected that a focus on conserved effectors will reveal insights that are applicable to the majority of oomycete pathogens. The investigators will perform a series of molecular genetic experiments to determine whether these effectors are capable of suppressing plant immunity or altering plant cell structure or physiology, and whether the pathogen's ability to cause disease is affected when these genes are silenced or overexpressed. Finally, one pair of conserved proteins will be selected for experiments to identify their exact targets inside plant cells. These experiments are expected to reveal important, broadly conserved molecular strategies that underpin oomycetes' ability to cause disease, which will in turn open new avenues toward more sophisticated disease control strategies. The project will provide multidisciplinary training for graduate students and undergraduates. Some aspects of the project will be collaboratively integrated with an outreach program at Virginia Tech that uses Arabidopsis reverse genetics in high school science classrooms.

Oomycete and fungal plant pathogens cause a number of very important diseases. Many of these pathogens can deliver proteins into the cytoplasm of their host cells to disable host immunity; these proteins are called effectors. The mechanism by which these proteins enter host cells is poorly understood. The investigators have shown that oomycete and fungal effector proteins can enter host cells without the aid of any pathogen-encoded machinery, but instead rely on short amino acid sequences in the N-termini called RXLR motifs. The investigators have discovered that the RXLR motifs can bind a class of membrane lipids called phosphoinositides. In this project, the investigators will test whether binding of the effectors to phosphoinositides is actually responsible for transferring the effectors into host cells. They will identify which host phosphoinositides are utilized by the effectors, and they will characterize the mechanism of binding. Finally they will test whether it is possible to block effector entry and so prevent disease. An extremely wide range of the most destructive plant pathogens, including those causing wheat rust, rice blast, grape powdery mildew and potato late blight depend on effectors. Understanding effector entry and determining how to block it would provide an entirely new approach to treating this broad class of diseases. Treatment of animal diseases caused by fungi, oomycetes and some parasites may also be treatable by this approach. This project will train two graduate students and provide summer research opportunities for multi-cultural undergraduate students.

PI: Brett Tyler (Virginia Polytechnic Institute and State University)

CoPIs: Shunyuan Xiao (University of Maryland Biotechnology Institute) and Mark Guiltinan (Pennsylvania State University)

Collaborator: Brian Bailey (USDA-ARS Sustainable Perennial Crops Lab, Beltsville, Maryland)

Small-holder agriculture in developing countries is exceptionally susceptible to fungal and oomycete disease due to lack of local breeding programs, tropical climate, abundant insect vectors, cost of and poor access to fungicides, and lack of farmer education. Small-holder farmers in Africa and South America are responsible for more than 95% of cacao production, providing economic benefits to millions of cacao farmers and their dependents, as well as important ecological benefits such as rain-forest preservation. Fungal and oomycete diseases have devastated cacao production in South America and West Africa, dislocating populations of farmers and resulting in destruction of the rain-forest sheltering the cacao trees in favor of large-scale open farmland. This project will test two novel approaches to protecting developing country crops against a broad-spectrum of oomycete and fungal pathogens, using cacao as the initial target. The first involves targeting anti-microbial proteins to pathogen feeding structures called haustoria, using RPW8, an Arabidopsis protein that has a natural affinity for these feeding sites. The second approach involves blocking the entry of virulence proteins (effector proteins) that pathogens secrete into host cells to suppress the plant host's immune system. The most effective strategies will be identified first using fast growing Arabidopsis and tomato plants before being tested in cacao.

Broader Impacts: The novel technologies that will be tested in this project are potentially applicable to an extremely broad array of crop diseases important to the developing world. The most destructive diseases of broad acreage staples such as rice and wheat are potentially amenable to this approach, as are many of the most limiting fungal and oomycete diseases of crops of importance to smallholders, such as yams, cassava, groundnuts, pearl millet, finger millet, sorghum, maize, potato, bean and cowpea. The project includes visits from scientists from developing countries, a colloquium in Ecuador, and regular communication with scientists from cacao-producing countries. Project outcomes as well as access to biological resources that include DNA constructs and germplasm will be disseminated through a project website (accessible via https://www.vbi.vt.edu/faculty/research_groups/brett_tyler) and through publications.

DESCRIPTION (provided by applicant): The inflammatory airway response in asthma may be the result of immune cells that are dysregulated towards environmental factors such as airborne fungi. The long-term goal of this project is to better understand the pathophysiological mechanisms of Th2-type airway inflammation in asthma. Exposure to the fungus, Alternaria, has long been implicated in the development and exacerbation of human asthma. After intranasal exposure to Alternaria spores and individual proteins, naive mice exhibit marked eosinophilic airway inflammation, enhanced Th2 sensitization to innocuous antigens, and airway hyperreactivity. Despite the well-documented clinical importance of Alternaria in the development, onset, and exacerbation of allergic airway diseases such as asthma, little knowledge exists about the role of individual fungal products in these pathological states. The importance of and detailed mechanisms of allergen uptake and movement through the epithelial cell layer (endocytosis and transcytosis) have not been explored to any great extent. This innovative project focuses on the secreted major Alternaria allergen Alt_a_1. It will assess the role of Alt_a_1 in allergic disease by utilizing bronchoalveolar epithelial cells and murine models of airway inflammation. We have discovered that Alt_a_1 is proinflammatory and harbors a unique amino acid motif (RXLR) that facilitates binding to cell surface PI-3-P and cellular entry. In aim 1 the role of Alt_a_1 in innate and adaptive immune responses in vitro and in murine models of asthma will be investigated. The effects of recombinant Alt_a_1 protein and fungal knockout mutants lacking Alt_a_1 will be examined. In specific aim 2 the importance of the Alt_a_1's esterase activity and RXLR motif in cellular entry and immunological responses will be characterized. Recombinant Alt_a_1 will be used to define amino acid residues required for esterase activity, for PI-3-P binding, and for cellular entry. Epithelial cell immune responses following treatment with the Alt_a_1 proteins will be investigated. This project will lead to a better understanding of the mechanisms of persistent and recurrent airway inflammation and may lead to the development of more specific and effective therapies and prevention strategies, especially if PI-3-P-mediated cell entry proves of general importance to allergenicity.

It is increasingly recognized that at the molecular level nonlinear networks of heterogeneous molecules control many plant processes, so that systems biology provides a valuable approach in this field, building on the integration of experimental biology with mathematical modeling. One of the biggest challenges to making this integration a reality is that most plant biologists do not possess the mathematical background needed to build and manipulate mathematical models well enough to use them as tools for hypothesis generation. The goal of the project is to develop a network modeling tool, PlantSimLab, that enables plant biologists to build, validate, and use intuitive computer models, with a user interface that does not require mathematical or modeling expertise. No such tool is currently available.

PlantSimLab is intended to play the role of a virtual laboratory, in which plant biologists can interrogate a computational model in ways similar to how it is done in the laboratory. The software will be developed in close collaboration with the plant biology community, through use cases that drive software capabilities and interface features. Finally, an extensive dissemination effort will be made to introduce the tool to plant biologists. The project will also leverage an ongoing outreach project that engages 9-12 year-old children across Virginia. The program, Kids' Tech University (KTU), brings 450 children to the Virginia Tech campus 4 times each year, where they interact with scientists, participate in hands-on laboratories, and, between campus events, explore a virtual space related to topics discussed on campus. The project will result in hands-on as well as virtual activities related to plant biology in general and the focus of the proposed project in particular.

An award is made to Oregon State University to purchase a confocal and two photon microscope system for life science research, education and training. Combined with the rapid increase in genomic information, confocal and two photon microscopy provides an unparalleled ability to identify the cellular components of living organisms, and to determine their functions. Key new features of this microscope include: two photon excitation for deep tissue imaging, and preservation of live organisms and cells; a tunable TiSa laser; 34 spectral detection channels; and high sensitivity GaAsP detectors for faster acquisition and dynamic imaging. Research and training programs of over 38 faculty in 12 departments and centers and 7 colleges will be strengthened and transformed by the novel capabilities of the new instrument. The research will advance knowledge of plants, animals, microbes, pathogens, symbionts and communities, in natural, managed, healthy and polluted environments, spanning agricultural, forest, ocean, and human ecosystems.

The novel capabilities of the two photon and confocal microscope, will advance solutions to the most complex challenges facing mankind including environmental health, ecosystem loss, sustainable food and energy production, infectious disease, and understanding behavior and the mind. A new undergraduate learning and research program on biological imaging will be enabled by the microscope acquisition and will provide students with the tools to understand and apply microscopy and imaging techniques, engage in inquiry-based research, conduct ethical and appropriate scientific methods. The interdepartmental Ph.D. program in Molecular and Cellular Biology program will be strengthened by the access provided students to cutting edge imaging technology. Access to this technology would benefit the broader Oregon research community, including small regional companies.

Oregon State University is upgrading key components of the campus core network infrastructure, enabling 10-40 Gbps network connections between research units within a Science DMZ, with interconnects via the DYNES network and Internet2 to external research partners such as XSEDE and iPlant. The upgrades include the campus core aggregation switch, two key data centers and six major data generating laboratories and facilities. Monitoring and optimization of overall network services within the upgraded DMZ includes implementation of perfSONAR for on- and off-campus performance monitoring. The DMZ creates a platform capable of integrating into Internet2 and utilizing DYNES and Software Defined Networking for the dynamic establishment of dedicated research network resources.

The upgrades transform research in a wide diversity of disciplines at Oregon State University (OSU) that generate and analyze massive data sets that are produced, analyzed and disseminated on- and off-campus. The disciplines affected include genomics and computational biology, image processing and recognition, earthquake and ocean wave physics, landscape remote sensing, pharmacy, clinical veterinary medicine, and earth, atmospheric and ocean sciences. OSU is a land grant, sea grant and sun grant institution. The network upgrades enhance its capability for providing solutions to society's problems in agriculture, aquaculture, forestry, fisheries and wildlife management, marine resource management, water quality management, environmental preservation and restoration, and animal and human health. Furthermore, the improved training and education environment that will be created by the upgrades positively impacts the professional development of graduate students, postdoctoral scholars and undergraduate researchers.

Beneficial and pathogenic microbes have major impacts on plants, animals, and humans, and on natural and managed ecosystems. Microbes that establish close interactions with plants and animals use a wide diversity of molecules to suppress or negotiate with their hosts' immune systems, including effector proteins that can enter host cells to directly re-program metabolism. Some microbes cause destructive diseases of plants, while other species provide major benefits. In all cases, it is poorly understood how effector proteins from microbes cross the membrane surrounding host cells to enter the cell interior. Although substantial progress has been made on this question, the technical challenges of the studies have slowed progress and have resulted in conflicting findings and controversy. This project will focus on solving the most severe technical challenges in this area, namely the question of how effector proteins interact with host membrane lipids, in order to reduce controversy and advance the field.

More specifically, the investigators will carry out detailed biochemical experiments aimed at resolving conflicting findings regarding the binding of oomycete effectors to the lipid phosphatidylinositol-3-phosphate. The work will focus on the two effectors from oomycetes for which the most structural and biochemical information is currently available, namely Phytophthora sojae Avh5 and Phytophthora capsici Avr3a4.

The project has three objectives.

1. Refine the effector protein expression and purification methods in order to identify the precise details required for obtaining preparations of the proteins that are active in host cell entry and lipid binding.

2. Identify at a very detailed level the biochemical conditions required to reproducibly observe binding of the effectors to phosphatidylinositol-3-phosphate.

3. In order to identify residues involved in binding, use precise and quantifiable methods to assay lipid binding by the purified effectors and their mutants, including Surface Plasmon Resonance and Isothermal Calorimetry, and circular dichroism spectroscopy.

In addition to advancing understanding of how microbes interact with plants, animals and humans, the broader impacts of this project will include the training of a postdoctoral scholar and several undergraduate researchers, and the mentoring of a junior investigator.

Understanding how the diversity of trees in forests is maintained is a global challenge. While the impact of invasive pests and new diseases caused by pathogens can be dramatic, the more subtle impacts of native pests and diseases are poorly understood. For example, can native pathogens and pests help promote (rather than destroy) biodiversity by limiting the most common tree species, creating room in the forest for rare species that would otherwise be outcompeted? This project will focus on the role of native pathogens, called oomycetes, in maintaining tree diversity in an old growth forest in the Pacific Northwest. Oomycetes, or water molds, are highly destructive fungus-like plant pathogens, best known for causing the Irish potato famine in the 1850s. Though well known as agricultural pathogens, oomycetes are native to and abundant in many forests. However, almost nothing is known about their ecological roles in natural forests. This project will (i) document the oomycete species present in the Wind River old growth forest in southern Washington state, (ii) determine the ability of each oomycete species to attack or limit different types of tree species in the forest, and (iii) use modern genetic techniques to explore how oomycetes may adapt to different types of tree species. By integrating all this information, the researchers will develop a better understanding of the roles of oomycetes and other pathogens in forests. This understanding will improve management of natural ecosystems and the diseases that occur within them. The project will also train undergraduate, graduate and postdoctoral students.

An initial census of plants and oomycetes, using both culturing and culture-independent methods, together with genotyping of the plants and oomycetes, will lead to plant-inoculation experiments that will identify the host ranges of the oomycetes. Genome and transcriptome sequencing will examine the mechanisms by which narrow host range oomycetes may adapt to genetic diversity in the dominant host plant species, and will examine more deeply how broad host range oomycetes may adapt to a diversity of plant species in their habitat. A particular focus will be on the role of endemic broad-host-range oomycetes, and how their interactions with dominant and non-dominant host species may differ. By integrating taxonomic, genetic, genomic and functional information about the oomycetes and plants at the Wind River site, two broad hypotheses regarding possible roles of oomycetes in influencing plant biodiversity will be tested. The two hypotheses are: (A) Narrow and broad host range pathogens may reduce the fitness of dominant hosts and so promote plant species and genetic diversity. (B) Dominant hosts may benefit from the presence of broad host range pathogens due to superior resistance or tolerance to broad-host-range pathogens. Both (A) and (B) may operate independently for different species, or even strains, of pathogens.

There is a growing awareness that the microbial populations that encompass them are an integral component of the life history of plants and the functioning of plant ecosystems. Understanding of how plants and their immune systems interact with pathogens and symbionts continues to rapidly advance. At the same time new understanding is emerging of how plants interact with broader populations of environmental microbes, especially resulting from the application of genomics, bioinformatics and systems biology. These new levels of understanding are leading to safer, more effective and more sustainable ways to manage plant pests and diseases in crops and natural ecosystems. The latest research findings on these topics will be presented and discussed at the 17th International Congress on Plant-Microbe Interactions in Portland, Oregon from July 17 through July 21, 2016. This grant will provide partial financial support for 20 promising U.S. students and postdoctoral scholars to attend this conference. These young scientists will participate in a small meeting workshop designed to enrich their engagement and participation. Established scientists and the supported participants will work in teams throughout the meeting to critically discuss the research findings. In addition, the students and postdocs will be contribute to the dissemination of the conference findings by releasing daily meeting highlights via social networking and publishing a final congress report.

Throughout its history since 1982, the International Congress on Plant-Microbe Interactions has been a unique and comprehensive congress covering both plant-pathogenic and plant-beneficial microbial interactions in both model and major crop plants, which distinguishes it from more specialized meetings. The 17th IC-MPMI will convene in Portland, Oregon from July 17 through July 21, 2016. In this meeting, the plenary and concurrent sessions will highlight the latest advances in fundamental research as well as translational studies critical for advancements in agricultural science. Highlights include a strong focus on the plant microbiome and multiway associations, a renewed focus on symbiotic associations, and new sessions on RNA-mediated interactions, population genomics, modeling and systems biology, and problems in the developing world. As with previous IC-MPMI congresses, the broader impacts of the 2016 congress will include the sharing of emerging data, the establishment and strengthening of international collaborations, and development of innovative strategies and technology to improve plant health, productivity and security. The participation of US scientists, especially graduate students and postdoctoral scholars, will be critical for the success and vitality of this congress. The workshop activities planned for the participants supported by this grant will enhance participation and learning by these young scientists, help them build their professional networks and communication skills, as well as inform others who are not able to attend this meeting.

Today, more than ever before, big data pervade every area of the life, environmental, biomedical, earth, marine, computational, physical, urban, and social sciences, as well as numerous other domains. Increasingly powerful computing technologies have opened the pathway for researchers to address major global challenges through use of large and heterogeneous data sets and through complex models and simulations. This project provides domain scientists, including research students, with the expertise and training needed to collaborate effectively with specialists in these advanced computational and statistical methodologies. The project also provides training and research experiences for students and instructors from small regional colleges, including Hispanic-serving and Native-American-serving institutions. To widely share best practices, it supports a community of practice.

The project employs research and training staff (facilitators) with expertise in data integration, multi-modal data analytics and machine learning. These three related sets of methods enable the analysis of large complex data sets of different types or from different sources, which may or may not have been collected as part of a planned studies. Specifically, a four-person facilitation team is established across Oregon State University, the University of Oregon, and Portland State University. The interdisciplinary, cross institutional team will establish the tools and managements practices to serve the researchers in the state. The research facilitated by this project will lead to better understanding of earthquakes, diverse ecosystems, and plant and animal form and function. It supports development of faster computing systems, more secure energy systems, and improved environmental health. The data challenges posed by these application areas also motivate new foundational research in advanced data analytics and machine learning. The project also prepares a new generation of students from diverse backgrounds to enter the knowledge economy.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.