Oomycetes are fungi-like eukaryotic microorganisms, which are actually phylogenetic relatives of diatoms and brown algae, within the kingdom Stramenopila. Many oomycete species, mainly in the genera Phytophthora, Pythium and downy mildews, are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Some oomycetes are also animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. Phytophthora species, represented by the Irish Potato Famine pathogen P. infestans and the soybean pathogen P. sojae, are arguably the most destructive pathogens of dicotyledonous plants among the oomycete species and thus have been extensively studied. This dissertation focuses on the model oomycete pathogen P. sojae to investigate specific aspects of its molecular biology and establish an efficient genetic manipulation tool.
Specifically, in Chapter 1, I briefly introduce the basic concepts of oomycete biology and pathology, and summarize the experimental techniques used for studies of oomycete genetics over the past two decades. Because the approach to studying fungi and oomycetes are similar (indeed they were incorrectly placed in the same taxonomic group until recently), a special section reviews the emerging genome editing technology CRISPR/Cas system in these organisms together.
Chapter 2 and Chapter 3 focus on one of the most important intracellular activities, nuclear localization of proteins, and describe the characterization of nuclear localization signals (NLSs) in P. sojae. This focus stemmed from my early work on genome editing in P. sojae, when I discovered that conventional NLS signals from SV40 used to target the TAL effector nuclease (TALEN) to the nucleus worked poorly in P. sojae. In the first part of this work (Chapter 2), I used confocal microscopy to identify features of nuclear localization in oomycetes that differ from animals, plants and fungi, based on characterization of two classes of nuclear localization signals, cNLS and PY-NLS, and on characterization of several conserved nuclear proteins. In the second part (Chapter 3), I determined that the nuclear localization of the P. sojae bZIP1 transcription factor is mediated by multiple weak nuclear targeting motifs acting together.
In Chapter 4 and Chapter 5, I describe my implementation of nuclease-based technology for genetic modification and control of P. sojae. In Chapter 4, I describe the first use of the CRISPR system in an oomycete, including its use to validate the function of a host specificity gene. This is of particular importance because molecular techniques such as gene knockouts and gene replacements, widely used in other organisms, were not previously possible in oomycetes. The successful implementation of CRISPR provides a major new research capability to the oomycete community. Following up on the studies described in Chapter 4, in Chapter 5, I describe the generalization and simplification of the CRISPR/Cas9 expression strategy in P. sojae as well as methods for mutant screening. I also describe several optimized methodologies for P. sojae manipulation based on my 5 years of experience with P. sojae. / Ph. D. / Oomycetes (water molds) are eukaryotic microorganisms that resemble filamentous fungi (molds), but are actually relatives of diatoms and brown algae, within a different kingdom of life named Stramenopila. The functional relationship between oomycetes and fungi is similar to that between fish and dolphins, which also acquired similar functions via different evolutionary paths. Many families of oomycetes are devastating plant pathogens that cause multibillion-dollar losses to agriculture annually in the world. Other families of oomycetes are animal pathogens, causing severe losses in aquaculture and fisheries, and occasionally causing dangerous infections of humans. <i>Phytophthora</i> species, represented by the Irish Potato Famine pathogen <i>P. infestans</i> and the soybean pathogen <i>P. sojae</i>, are among the most destructive oomycete pathogens of plants and thus have been extensively studied. This dissertation is focused on the model oomycete pathogen <i>P. sojae</i>. It investigates specific aspects of its molecular biology and establishes an efficient genetic manipulation tool. All complex organisms (eukaryotes) package their genetic material in nuclei, which contain proteins as well as DNA. In the first part of my research (Chapter 2 and Chapter 3), I focused on the mechanisms used by <i>P. sojae</i> to target nuclear proteins into the nucleus, particularly the tags (called nuclear localization signals, or NLSs) that are identify the proteins that must travel to the nucleus. I showed that nuclear targeting mechanisms in oomycetes differ in distinct ways from well-studied eukaryotes such as humans. In particular, the nuclear targeting signals in <i>P. sojae</i> proteins are diffused over multiple sites on the proteins, whereas in human proteins there’s usually just a single signal. For one particular oomycete protein, a transcription factor, nuclear targeting involves four weak signals that cooperate synergistically. Two of these four weak signals define a new class of nuclear localization signal. In the second part of my research (Chapter 4 and Chapter 5), I implemented and further optimized a genome editing technology for genetic modification and control of <i>P. sojae</i>. This technology is based on the CRISPR system that has revolutionized genome editing in plants and animals over the last three years. This is of particular importance because genome editing techniques were not previously possible in oomycetes. The successful implementation of CRISPR technology in <i>P. sojae</i> has provided a major new research capability to the oomycete community. In Chapter 5, I also describe several optimized methodologies for <i>P. sojae</i> genetic manipulation based on my 5 years of experience with <i>P. sojae</i>.
| Identifer | oai:union.ndltd.org:VTETD/oai:vtechworks.lib.vt.edu:10919/74232 |
| Date | 15 November 2016 |
| Creators | Fang, Yufeng |
| Contributors | Animal and Poultry Sciences, Tyler, Brett M., McDowell, John M., Lawrence, Christopher B., Freitag, Michael |
| Publisher | Virginia Tech |
| Source Sets | Virginia Tech Theses and Dissertation |
| Detected Language | English |
| Type | Dissertation |
| Format | ETD, application/pdf, application/octet-stream, application/pdf |
| Rights | In Copyright, http://rightsstatements.org/vocab/InC/1.0/ |
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