The Microbotryum violaceum fungal complex is an exciting system for examination of the ecology and evolution of host/pathogen interactions and can serve as a model for emerging infectious diseases in 'wild' non-agricultural environments where host heterogeneity provides an important contrast to heavily studied agricultural pathogens. Current genomics scale high-throughput technologies are invaluable for gaining insight into the complex systems and associated interactions that result in disease progression. This project will provide a genomics infrastructure necessary for the rational design of hypothesis driven inquiries into M. violaceum development and pathogenesis. A comprehensive and comparative view will be generated of the suites of genes that play significant roles in fungal development and successful infection. The specific objectives are to: (1) Sequence and assemble the complete genome of Microbotryum violaceum, which is estimated to be 25 Mb; (2) Annotate gene structure using computational methods; (3) Analyze gene transcription profiles late in infection; (4) Promptly release publicly all reads, assemblies, and annotation; and (5) Develop education, training and outreach programs. Broader impacts: Several research groups are prepared to use the genome sequence to analyze signaling leading to fungal development and disease production, host specificity, genes under positive selection, evolution of sex, and gene expression during infection. A website will be developed at the Broad Institute to disseminate the information on the genome of M. violaceum as well as allow comparison to other genomes, including Ustilago maydis, the causative agent of corn smut disease. These resources will be used by a community of scientists in the public and private sectors interested in basic fungal biology, host-parasite interactions, fungal evolution and the development of new methods for protecting agricultural crop plants from fungal diseases through a better understanding of the basic infection mechanism and disease process of obligate plant parasitic fungi. Furthermore, this project will provide educational opportunities for undergraduate students to participate in fungal genomics and bio-informatics research and broaden the participation in basic science by under-represented groups (specifically, African-Americans, women, Appalachians). An outreach mechanism will enhance the interest of such undergraduates in pursuing an advanced degree in the biological sciences.
Intellectual Merit: The main goals of this project were to determine and characterize the genomic sequence for a fungal plant pathogen, Microbotryum violaceum, and to provide this information to the research community on a publicly-available database. The genome sequence shows that there is a relatively large amount of repetitive DNA sequence for a comparatively small genome. Moreover, the genome sequence allowed us to characterize the "sex chromosome" of this fungus, the chromosome that contains the genes controlling its mating process (See "Mating Chromosome" image). In addition, we were interested in examining "total" gene expression for the organism during a wide variety of stages during its lifecycle (Figure 1). These included several stages of its lifecycle outside of its plant host, Silene latifolia, a wildflower species in the carnation family; in addition, we did a careful analysis of gene expression during the late stages of infection, when the fungus replaces the pollen of its host plant with its own fungal spores. This last stage of infection is important, since it is here that the fungus undergoes a final developmental switch, from a filamentous form to a diploid spore form. The gene expression analyses have identified a number of potentially interesting genes that appear to encode small proteins secreted by the fungus; these have the hallmarks of "effectors" used by fungi to control the development and behavior of their host plants during infection. Finally, the information from these studies has allowed us to develop a reliable method to further study this fungus via a genetic manipulation known as "transformation." These findings have been published in two scientific journal articles and have resulted in 4 presentations at international scientific conferences. Moreover, two additional articles have already been favorably reviewed and are in revision; another is ready to submit and two others are in preparation. Broader Impacts: This project has provided a publicly-available database that will be used by the community of researchers studying the evolution and molecular biology of fungal/plant interactions. Its findings have important implications for the evolution of sex chromosomes and the evolution of intracellular parasites, such as "selfish" DNAs (transposable elements). Overall, this project has supported the training of 1 faculty member at a regional university, 4 graduate students, and 15 undergraduates. Among all these trainees, 13 were female, 3 were African-American, 8 were Asian or Asian-American, and 1 was from Appalachia. Outreach: One of our summer undergraduate students in the lab (Heather Meisch, 2010) was recruited from Berea College, a school that only accepts economically disadvantaged students, typically from Appalachia or from under-represented groups, such as African Americans, Native Americans, or Hispanics. Brittney Hubert, a Brown Scholar at the University of Louisville, worked in our lab during the academic year. She also spent summer, 2011, at the Universite de Paris-Sud, in Orsay, France, with our collaborator, Dr. Tatiana Giraud. There she obtained additional practical training with the fungus and with growth and infection of the S. latifolia host. This internship is also part of our efforts to solidify the ongoing and future collaborative efforts between our two labs. A currnt continuing student, Ann-Claude Rakotoniaina, will also be visiting the lab of Dr. Giraud this summer. We continue our efforts to inform and recruit students from Kentucky and the surrounding region, by our ongoing programs with Kentucky State University, Berea College, and Bennett College. As part of this ongoing effort, in October, 2012, the PI visited Centre College in Danville, KY, to present a seminar to undergraduates on the current status of our findings for this NSF project and their significance.
