Browsing by Subject "transcriptome"

The kingdom Fungi is one of the major groups of the plant microbiome(Hardoim et al., 2015; Vandenkoornhuyse et al., 2015; Peay et al., 2016). Of the various plant-fungus interactions, mycorrhizal fungi that form mutualistic associations with host plants are the best studied symbiotic system(Bonfante & Genre, 2010; van der Heijden et al., 2015). Fungal endophytes represent another major type of plant-fungus symbioses(Rodriguez et al., 2009; Porras-Alfaro & Bayman, 2011). Defined as endosymbionts inhabiting a wide range of plant and lichen hosts without causing obvious symptoms, endophytes are now considered both ubiquitous and hyperdiverse (Stone, 2004; Rodriguez et al., 2009; U'Ren et al., 2012). Yet most of these fungi have to be identified using a phylogenetic approach (Arnold et al., 2009; Gazis et al., 2012; Chen et al., 2015) and remain unknown at lower taxonomic ranks (e.g., genus and species) and undefined in terms of their function in their symptomless hosts(Arnold et al., 2003; Busby et al., 2016). It is now understood that some endophytes are capable of switching to pathogenic(Wipornpan Photita et al.; Ávarez-Loayza et al., 2011) or saprotrophic(U'Ren et al., 2010; Zuccaro et al., 2011; Kuo et al., 2014) modes, but the genetic mechanisms of these switches remain unexplored. Bryophytes are a major component of the vegetation in boreal and arctic regions, where ecosystems are most vulnerable to global climate change(Turetsky et al., 2012; Jassey et al., 2013). It has been proposed that early land plants adopted a terrestrial lifestyle with the help of fungi(Heckman et al., 2001; Field et al., 2015). Mosses do not have mutualistic fungal symbionts such as mycorrhizal fungi(Davey & Currah, 2006; Field et al., 2015), but they are known to harbor diverse fungal endophytes of uncertain functions(U'Ren et al., 2010; Davey et al., 2012; Davey et al., 2013). The growth form of the moss Dicranum scoparium provided an ideal system for studying functional transitions between endophytism and saprotrophism across a senescent gradient. My PhD thesis focuses on the evolutionary history (Chapter 1) and functionality (Chapter 2, 3) of endophytic fungi.

In Chapter 1, I investigated the phylogenetic placements of fungal endophytes within the pharmaceutically and agriculturally important class Eurotiomycetes. The class Eurotiomycetes (Pezizomycotina, Ascomycota) includes various fungi with different ecological traits, including animal pathogens, saprotrophs, ectomycorrhizae, plant pathogens, rock-inhabiting fungi, lichens and endophytes(Geiser et al., 2006; Schoch et al., 2009; Gueidan et al., 2015). Phylogenetic affiliations of eurotiomycetous fungal endophytes with their ecologically diverse relatives had not been evaluated, leaving a gap in our understanding of the major evolutionary trends and ecological breadth of Eurotiomycetes as a whole. To fill this gap, we recently inferred the phylogenetic and taxonomic affinities of representatives of class 3 endophytes within Eurotiomycetes (Chen et al., 2015). Our results based on seven loci and 157 taxa revealed an undescribed new order (Phaeomoniellales) composed mainly of fungal endophytes and plant pathogens, and to a lesser extent, endolichenic and lichen-forming fungi. However, most of the deep nodes within this order were poorly supported. Interestingly, while described species of the order Phaeomoniellales are mostly plant pathogens on angiosperms (e.g., Genera Vitis, Nephelium and Prunus(Groenewald et al., 2001; Damm U. et al., 2010; Rossman et al., 2010; Thambugala et al., 2014)), endophytes within this order were mostly isolated from leaves of gymnosperms (Fig.1). These results, first-authored by the Co-PI, have been published in the journal Molecular Phylogenetic and Evolution(Chen et al., 2015).

In Chapter 2, I used metatranscriptomes of fungal ribosomal RNA to detect active fungal communities across a gradual gradient of senescence in wild-collected gametophytes of Dicranum scoparium (Bryophyta) to understand the distribution and the active component of fungal communities at a given time in adjacent living, senescing, and dead tissues. My results suggested that Ascomycota generally were more prevalent and active in living tissues, whereas Basidiomycota were more prevalent and active in senescing and dead tissues. Differences in community assembly detected by metatranscriptomics were echoed by amplicon sequencing of cDNA and compared to culture-based inferences and observation of fungal fruit bodies in the field. The combination of metatranscriptomics and amplicon sequencing of cDNA is promising for studying symbiotic systems with complex microbial diversity, allowing simultaneous detection of microbial presence, abundance and metabolic activity in symbiotic systems.

In Chpater3, I investigated the functions of D. scoparium across its naturally occurring senescence gradient and the associated fungal nutrient transporter (carbon, amino acid, phosphorus and nitrogen) activities. Higher fungal nutrient-related transporter activities were detected toward the bottom layer of the moss gametophytes. Among the four fungal nutrient types (Amino acid, carbon, nitrogen, phosphorus), the activities of nitrogen-related transporters had a drastic increase proportionally toward the bottom layer. In parallel, nitrogen breakdown was detected as the most enriched Gene Ontology term of D. scoparium for those transcripts having higher expression in the bottom layers. I analyzed the most abundant fungal nitrogen-related transporters in my dataset, the ammonium transporters, using a phylogenetic approach. I revealed that all ammonium transporters actively expressed in association with D. scoparium belong to the MEPg clade. Different sets of potential plant-microbe communication/defense/symbiosis-related genes are highly expressed in top vs. bottom layers, which suggest different mechanisms are involved in plant-fungus associations in photosynthetic vs. decomposing tissues.

This research examines three potential mechanisms by which bacteria can adapt to different temperatures: changes in strain-level population structure, gene regulation and particle colonization. For the first two mechanisms, I utilize bacterial strains from the Vibrionaceae family due to their ease of culturability, ubiquity in coastal environments and status as a model system for marine bacteria. I first examine vibrio seasonal dynamics in temperate, coastal water and compare the thermal performance of strains that occupy different thermal environments. Our results suggest that there are tradeoffs in adaptation to specific temperatures and that thermal specialization can occur at a very fine phylogenetic scale. The observed thermal specialization over relatively short evolutionary time-scales indicates that few genes or cellular processes may limit expansion to a different thermal niche. I then compare the genomic and transcriptional changes associated with thermal adaptation in closely-related vibrio strains under heat and cold stress. The two vibrio strains have very similar genomes and overall exhibit similar transcriptional profiles in response to temperature stress but their temperature preferences are determined by differential transcriptional responses in shared genes as well as temperature-dependent regulation of unique genes. Finally, I investigate the temporal dynamics of particle-attached and free-living bacterial community in coastal seawater and find that microhabitats exert a stronger forcing on microbial communities than environmental variability, suggesting that particle-attachment could buffer the impacts of environmental changes and particle-associated communities likely respond to the presence of distinct eukaryotes rather than commonly-measured environmental parameters. Integrating these results will offer new perspectives on the mechanisms by which bacteria respond to seasonal temperature changes as well as potential adaptations to climate change-driven warming of the surface oceans.

Cell invasion through basement membrane (BM) barriers is necessary for development and homeostasis, and is misappropriated in diseases like cancer. Many regulators of this complex biological process have been identified by relying on studies completed in vitro and through the analysis of genetic mutants in vivo, however, these methods are unable to identify redundant and subtle regulators of invasion. Capturing the gene expression profile of a cell actively transmigrating BM in vivo remains elusive as invasion through BM is often random. However, a gene expression profile would shed light on genes and pathways that have previously gone unnoticed using traditional screening methods. Here, I use the C. elegans model of anchor cell (AC) invasion through BM as a visually and genetically tractable in vivo model utilizing forward and reverse genetic screening, transcriptome profiling, split-GFP protein tagging strategies, and live cell imaging to investigate and identify new regulators and cellular processes controlling cell invasion through BM. In Chapter 1, I review strategies and mechanisms that invasive cells employ to breach and travel through BM and present AC invasion in C. elegans as an in vivo model of cell invasion. In Chapter 2, I attempt to identify two new regulators of AC invasion identified through a large-scale forward genetic screen. In Chapter 3, by developing methods to isolate individual AC’s, I identify the AC transcriptional profile during invasion, I reveal key roles for ribosome biogenesis and endomembrane expansion in cell invasion through BM and establish the AC transcriptome as a resource to identify mechanisms underlying BM transmigration. Chapter 4 discusses the implications of these findings and future directions.