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Browsing by Author "Fang, Yufeng"

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    Distinctive Nuclear Localization Signals in the Oomycete Phytophthora sojae
    Fang, Yufeng; Jang, Hyo Sang; Watson, Gregory W.; Wellappili, Dulani P.; Tyler, Brett M. (Frontiers, 2017-02-02)
    To date, nuclear localization signals (NLSs) that target proteins to nuclei in oomycetes have not been defined, but have been assumed to be the same as in higher eukaryotes. Here, we use the soybean pathogen Phytophthora sojae as a model to investigate these sequences in oomycetes. By establishing a reliable in vivo NLS assay based on confocal microscopy, we found that many canonical monopartite and bipartite classical NLSs (cNLSs) mediated nuclear import poorly in P. sojae. We found that efficient localization of P. sojae nuclear proteins by cNLSs requires additional basic amino acids at distal sites or collaboration with other NLSs. We found that several representatives of another well-characterized NLS, proline-tyrosine NLS (PY-NLS) also functioned poorly in P. sojae. To characterize PY-NLSs in P. sojae, we experimentally defined the residues required by functional PY-NLSs in three P. sojae nuclear-localized proteins. These results showed that functional P. sojae PY-NLSs include an additional cluster of basic residues for efficient nuclear import. Finally, analysis of several highly conserved P. sojae nuclear proteins including ribosomal proteins and core histones revealed that these proteins exhibit a similar but stronger set of sequence requirements for nuclear targeting compared with their orthologs in mammals or yeast.
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    Diverse Evolutionary Trajectories for Small RNA Biogenesis Genes in the Oomycete Genus Phytophthora
    Bollmann, Stephanie R.; Fang, Yufeng; Press, Caroline M.; Tyler, Brett M.; Gruenwald, Niklaus J. (Frontiers, 2016-03-15)
    Gene regulation by small RNA pathways is ubiquitous among eukaryotes, but little is known about small RNA pathways in the Stramenopile kingdom. Phytophthora a genus of filamentous oomycetes, contains many devastating plant pathogens, causing multibillion-dollar damage to crops, ornamental plants, and natural environments. The genomes of several oomycetes including Phytophthora species such as the soybean pathogen P. sojae, have been sequenced, allowing evolutionary analysis of small RNA-processing enzymes. This study examined the evolutionary origins of the oomycete small RNA-related genes Dicer-like (DCL), and RNA-dependent RNA polymerase (RDR) through broad phylogenetic analyses of the key domains. Two Dicer gene homologs, DCL1 and DCL2, and one RDR homolog were cloned and analyzed from P sojae. Gene expression analysis revealed only minor changes in transcript levels among different life stages. Oomycete DCL1 homologs clustered with animal and plant Dicer homologs in evolutionary trees, whereas oomycete DCL2 homologs clustered basally to the tree along with Drosha homologs. Phylogenetic analysis of the RDR homologs confirmed a previous study that suggested the last common eukaryote ancestor possessed three RDR homologs, which were selectively retained or lost in later lineages. Our analysis clarifies the position of some Unikont and Chromalveolate RDR lineages within the tree, including oomycete homologs. Finally, we analyzed alterations in the domain structure of oomycete Dicer and RDR homologs, specifically focusing on the proposed domain transfer of the DEAD-box helicase domain from Dicer to RDR. Implications of the oomycete domain structure are discussed, and possible roles of the two oomycete Dicer homologs are proposed.
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    Nuclear Localization of Proteins and Genome Editing in the Oomycete Phytophthora sojae
    Fang, Yufeng (Virginia Tech, 2016-11-15)
    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.