Magnaporthe grisea is a notorious pathogenic fungus that causes rice blast disease worldwide. Proteins secreted by the fungus are likely candidates for being effectors that are potentially recognized by determinants of resistance or susceptibility in host plants. However, knowledge of the role of secreted proteins of M. grisea is still limited. In this study, I identified 29 proteins that were secreted into culture filtrates from M. grisea strains expressing candidate proteins. I confirmed secretion of these proteins and tested them for elicitor activity on plants. Among them, I studied two groups: cell wall degrading enzymes (CWDEs) and small cysteine-rich proteins. Cysteine-rich proteins have been shown in other systems to function as elicitors. Initially, I expressed and purified proteins in M. grisea to obtain proteins by a homologous expression system. Although this was effective for a number of proteins, the need for greater amounts of protein led me to express several proteins in the Pichia pastoris system. Several candidate proteins were purified and found to induce symptoms on rice and maize. Hypothetical proteins MG10424.4 and MG09998.4 were both found to have elicitor activity. Lipase MG07016.4 did not induce response of plants and we concluded that the lipase activity of MG07016.4 does not function as an elicitor. I also purified a small cysteine-rich protein, which belongs to the group of cluster 180 proteins in M. grisea, MG10732.4 from P. pastoris. It is able to cause yellowing symptoms and hydrogen peroxide production in plants and it might contain elicitor activity.

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The numerous benefits associated with natural products isolated from the environmental sources, including soil bacteria, plants and fungi, are long known and well appreciated. Interestingly, the immense number of microorganisms that reside within our bodies and whose cell counts greatly outnumber our own represents a potentially new and practically untapped reservoir of bioactive compounds. With the advent of next generation sequencing we are only now starting to realize the complexity and biological diversity of the human microbiome. With this ever-increasing flow of genomic information, more bioactive potential in these microbes can be identified. For instance, biosynthetic assembly lines responsible for the production of two largest classes of bioactive compounds, polyketides and nonribosomal peptides, can be readily identified within the microbial genomes, providing us with a view of their bioactive profiles. In addition to the identification of biosynthetic assembly lines, the building blocks of polyketide and nonribosomal peptide products can also be accurately predicted, given the well-understood logic of assembly line operations. Nonetheless, the identification of actual products is still lagging behind. The discovery of these bioactive molecules can be achieved, however, by establishing a unique connection between genomes and molecules. Using several concrete examples, this thesis demonstrates how both metabolomic and biochemoinformatic platforms can assist in discovery of bioactive small molecules. More specifically, investigations involving three members of the human microbiome, Streptococcus mutans, Lactobacillus plantarum and Pseudomonas aeruginosa, provide distinct examples of identification of bioactive agents and assessment of their immunomodulatory potential. Interrogating the human microbiome form the angle of small molecules is critical for evaluation of microbial effects on our cells, and ultimately our health. Studying these agents will hopefully reveal interesting principles on how microorganisms speak to human cells and how this communication could lead to therapeutic strategies or downstream mechanistic revelations. / Thesis / Master of Science (MSc) / The numerous benefits associated with natural products isolated from the environmental sources, including soil bacteria, plants and fungi, are long known and well appreciated. Interestingly, the immense number of microorganisms that reside within our bodies and whose cell counts greatly outnumber our own represents a potentially new and practically untapped reservoir of bioactive compounds. With the advent of next generation sequencing we are only now starting to realize the complexity and biological diversity of the human microbiome. With this ever-increasing flow of genomic information, more bioactive potential in these microbes can be identified. For instance, biosynthetic assembly lines responsible for the production of two largest classes of bioactive compounds, polyketides and nonribosomal peptides, can be readily identified within the microbial genomes, providing us with a view of their bioactive profiles. In addition to the identification of biosynthetic assembly lines, the building blocks of polyketide and nonribosomal peptide products can also be accurately predicted, given the well-understood logic of assembly line operations. Nonetheless, the identification of actual products is still lagging behind. The discovery of these bioactive molecules can be achieved, however, by establishing a unique connection between genomes and molecules. Using several concrete examples, this thesis demonstrates how both metabolomic and biochemoinformatic platforms can assist in discovery of bioactive small molecules. More specifically, investigations involving three members of the human microbiome, Streptococcus mutans, Lactobacillus plantarum and Pseudomonas aeruginosa, provide distinct examples of identification of bioactive agents and assessment of their immunomodulatory potential. Interrogating the human microbiome form the angle of small molecules is critical for evaluation of microbial effects on our cells, and ultimately our health. Studying these agents will hopefully reveal interesting principles on how microorganisms speak to human cells and how this communication could lead to therapeutic strategies or downstream mechanistic revelations.

Musculoskeletal diseases, in particular osteoporosis, are increasingly becoming more prevalent in the U.S. due to the ageing population (Figure1). It is estimated that one-sixth of 300 million people in U.S. suffer from bone disorders or loss. About 10 million of those people above age 50 suffer from osteoporosis. Patients that suffer from osteoporosis have high morbidity and mortality rates. For instance, patients have decreased bone mineral density (BMD), a measurement of bone density that reflects the strength of bone as represented by calcium content. A decrease in BMD typically leads to an increased risk of bone fractures. In particular, hip fractures have an associated 20% mortality rate 1 year after injury among senior citizens 1. Patients that suffer from musculoskeletal diseases and from bone injuries, not associated with disease, account for 130 million hospital visit per year. Not to mention, 245 billion dollars of healthcare expenditure 2. Over that last 30 years, there has been much improvement in the field of bone research and its application to medicine. It has changed the quality of life and prolonged the life expectancy of patients suffering from bone disease. However, many details remain unknown about the underlying mechanism that control bone metabolism, formation, and healing. Furthermore, current effective therapies to combat bone disorders have limitations including unwanted side effects and prohibitive costs. For example, treatment with glucocorticoids which is a known inducer of osteoblastogenesis in vitro has been shown to produce an osteoporotic phenotype in vivo. Recognizing the importance of bone health and its affordability to the public makes the advancement of therapeutic targets work worth doing. Work in this field will eventually lead to the prevention, treatment, and cure for bone disease. A potential therapeutic candidate that maybe involved directly or indirectly with bone formation is secreted phosphoprotein-24 (Spp24). The following research aims to establish an importance and role for Spp24 in bone differentiation. A novel antibody that detects Spp24 which we have developed and characterized, has allowed us to feasibly study the protein. Our results demonstrate localization of Spp24 in different tissue, the processing of the protein during osteoblastogenesis, and have allowed us to conceptualize possible functions based on our data.

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Les protéases Tolloïdes constituent un groupe de métalloprotéases extracellulaires comptant quatre membres chez les mammifères (BMP-1, mTLD, mTLL-1 et mTLL-2). Ces protéases jouent un rôle majeur dans le développement et la réparation tissulaire, ainsi que dans certaines pathologies comme les fibroses. En 2006, le premier inhibiteur endogène des protéases Tolloïdes a été identifié chez le xénope et le poisson zèbre. Il s'agit de la protéine Sizzled, qui appartient à la famille des secreted Frizzled-Related proteins (sFRPs). Le travail présenté dans ce manuscrit suggère que ce mécanisme d'inhibition des protéases Tolloïdes par les sFRPs n'est pas conservé chez les mammifères. En effet, trois des cinq sFRPs de mammifères ont été testées (sFRP1, sFRP2 et sFRP4), et aucune d'entre elles ne s'est avérée capable d'inhiber l'activité de la protéase BMP-1 humaine in vitro. Ce travail montre toutefois que les protéases BMP-1, mTLD et mTLL-1 humaines peuvent être inhibées de façon puissante et spécifique par la protéine Sizzled de xénope. Cette inhibition repose sur l'interaction du domaine Frizzled de Sizzled avec le domaine catalytique des protéases Tolloïdes. Plus particulièrement, les résidus Asp-92, Phe-94, Ser-43 et Glu-44 de Sizzled (dont certains ne sont pas présents chez les sFRPs de mammifères) jouent un rôle crucial dans cette inhibition. Enfin, nous nous sommes intéressés au variant long du collagène XVIII, qui comporte également un domaine Frizzled. Nous avons pu montrer que BMP-1 clive le collagène XVIII in vitro, libérant un fragment contenant le domaine Frizzled. Des expériences sont en cours pour déterminer si ce fragment est capable d'inhiber les protéases Tolloïdes / Tolloid proteinases constitute a group of extracellular metalloproteinases which includes four members in mammals (BMP-1, mTLD, mTLL-1, mTLL-2). These proteinases play major roles in development, tissue repair and related pathological conditions such as fibrosis. In 2006, the first endogenous inhibitor of Tolloid proteinases was identified in Xenopus and zebrafish. This inhibitor, called Sizzled, is a member of the secreted Frizzled- related proteins (sFRPs). The present study strongly suggests that inhibition of Tolloid proteinases activity by sFRPs is not conserved in mammals. Indeed, three of the five mammalian sFRPs were tested (sFRP1, sFRP2 and sFRP4) and none of them was found to inhibit human BMP-1 activity in vitro. In contrast, this study demonstrates that Xenopus Sizzled is a potent and specific inhibitor of human BMP-1, mTLD and mTLL-1. This inhibition involves an interaction between the Frizzled domain of Sizzled and the catalytic domain of Tolloid proteinases. More precisely, residues Asp-92, Phe-94, Ser-43 and Glu-44 of Sizzled (among which only Asp-92 is conserved in mammalian sFRPs) play a crucial role in Tolloid proteinase inhibition. Finally, we studied the longest isoform of collagen XVIII, which also contains a Frizzled domain. We found that BMP-1 can cleave collagen XVIII in vitro, resulting in a Frizzled domain-Containing fragment. Experiments are in progress to determine if this fragment can also inhibit Tolloid proteinase activity

Doctor of Philosophy / Department of Plant Pathology / John P. Fellers / Harold Trick / In Kansas, wheat (Triticum aestivum L.) is severely affected by the biotrophic fungus Puccinia triticina (leaf rust). Although resistant varieties have been developed, the fungus tends to overcome new sources resistance very quickly. Plants have evolved a single gene (R genes) defense network that can recognize specific pathogen effectors (Avr), in a gene-for-gene manor. In rusts, effectors are secreted proteins responsible for inducing the uptake of nutrients and inhibit host defense responses. Identification of secreted proteins during the infection may help to understand the mode of infection of P. triticina. Little is known about molecular interactions in the pathosystem wheat-leaf rust and no Avr genes from cereal rusts have been cloned. In order to understand pathogenicity in leaf rust and generate new alternatives for disease control, the goal of this research is identify P. triticina secreted proteins from a collection of expressed genes during the infection, and to characterize putative Avr function for three candidates. From 432 EST’s derived from haustoria and infected plants, fifteen secreted proteins were identified and 10 were selected as potential avirulence candidates. Pt3 and Pt 51 are two P. triticina (Pt) candidates expressed specifically in the haustoria and encode small cysteine-rich secreted proteins. Eight candidates are expressed at early stages of infection, during spore germination and 6 days after inoculation. They are small-secreted proteins. None are repetitive elements or have nuclear localization signals. They also do not share a conserved motif with known filamentous fungus Avr proteins. Five candidates are novel proteins, two have similarity with predicted proteins, one is homologous with Hesp-379-like protein, one is homologous with superoxide dismutase, and one has a cell glucanase predicted function. Pt3, Pt12 and Pt27 were tested by transient expression experiments using co-bombardment with GUS into leaf rust resistant isogenic lines. Reduction in the expression of reporter gene GUS co-expressed with Pt27 indicates a potential avirulence factor for Lr26 in wheat.

Doctor of Philosophy / Department of Plant Pathology / Barbara S. Valent / Rice blast caused by the ascomycetous fungus Magnaporthe oryzae remains a threat to global sustainable agriculture and food security. This pathogen infects staple cereal crops such as rice, wheat, barley and millets, as well as turf grasses, in a distinct way among fungal plant pathogens, which we described in the first chapter. In addition to economical importance, rice blast is a model pathosystem for difficult-to-study biotrophic fungi and fungal-plant interactions. We are studying proteins that fungi secrete inside living cells to block plant defenses and control host cell processes; these proteins are called effectors. To date mechanisms for secretion and delivery of effectors inside host cells during disease establishment remain unknown. This step is critical to ensure the successful infection. So far, the only commonality found among all unique small-secreted blast effector proteins is their accumulation in a novel in planta structure called the biotrophic-interfacial complex (BIC). Identifying effectors and understanding how they function inside rice cells are important for attaining durable disease control. In the second chapter, we presented one approach to address this challenge. We characterized four candidate effector genes that were highly expressed specifically during the rice cell invasion. Using transgenic fungi that secrete fluorescently-labeled versions of each protein allowed me to follow them during invasion in vivo by live cell imaging. These candidates show distinct secretion patterns suggesting a spatially-segregated secretion mechanism for effectors. Results revealed a BIC-located strong candidate cytoplasmic blast effector, two putative cell-to-cell movement proteins and a putative extrainvasive hyphal membrane (EIHM)-matrix protein, which has become a valuable tool for assessing successful infection sites. In the third chapter, we test if normal secretion components of filamentous fungi are involved in accumulation of effectors into BICs. We report localization studies with M. oryzae orthologs of conserved secretion machinery components to investigate secretion mechanisms for effectors showing preferential BIC accumulation and for non-BIC proteins such as BAS4. Especially bright fluorescence adjacent to BICs from Mlc1p (Myosin Light Chain, a Spitzenkörper marker), from Snc1p (a secretory vesicle marker), and from Yup1p (a putative t-SNARE endosomal protein) suggest secretion actively occurs in the BIC-associated cells. Localization of Spa2p (a polarisome marker), as a distinct spot at the tips of the bulbous invasive hyphae (IH) in planta, suggests the existence of two secretion complexes after the fungus switches growth from the polarized filamentous primary hyphae to bulbous IH. In the final chapter on future perspectives, we present some strategies towards the molecular understanding of the M. oryzae secretion mechanism during biotrophic invasion, which will lead to novel strategies for disease control.