Browsing by Author "Lutzoni, François"

Although common knowledge dictates that the lichen thallus is formed solely by a fungus (mycobiont) that develops a symbiotic relationship with an alga and/or cyanobacterium (photobiont), the non-photoautotrophic bacteria found in lichen microbiomes are increasingly regarded as integral components of lichen thalli and significant players in the ecology and physiology of lichens. Despite recent interest in this topic, the phylogeny, ecology, and function of these bacteria remain largely unknown. The experiments presented in this dissertation employ culture-free methods to examine the bacteria housed in these unique environments to ultimately inform an assessment of their status with regard to the lichen symbiosis. Microbiotic surveys of lichen thalli using new oligonucleotide-primers targeting the 16S SSU rRNA gene (developed as part of this study to target Bacteria, but exclude sequences derived from chloroplasts and Cyanobacteria) revealed the identity of diverse bacterial associates, including members of an undescribed lineage in the order Rhizobiales (Lichen-Associated Rhizobiales 1; `LAR1'). It is shown that the LAR1 bacterial lineage, uniquely associated with lichen thalli, is widespread among lichens formed by distantly related lichen-forming fungi and is found in lichens collected from the tropics to the arctic. Through extensive molecular cloning of the 16S rRNA gene and 454 16S amplicon sequencing, ecological trends were inferred based on mycobiont, photobiont, and geography. The implications for using lichens as microcosms to study larger principles of ecology and evolution are discussed. In addition to phylogenetic and ecological studies of lichen-associated bacterial communities, this dissertation provides a first assessment of the functions performed by these bacteria within the lichen microbiome in nature through 454 sequencing of two different lichen metatranscriptomes (one from a chlorolichen, Cladonia grayi, and one from a cyanolichen, Peltigera praetextata). Non-photobiont bacterial genes for nitrogen fixation were not detected in the Cladonia thallus (even though transcripts of cyanobacterial nitrogen fixation genes from two different pathways were detected in the cyanolichen thallus), implying that the role of nitrogen fixation in the maintenance of chlorolichens might have previously been overstated. Additionally, bacterial polyol dehydrogenases were found to be expressed in chlorolichen thalli (along with fungal polyol dehydrogenases and kinases from the mycobiont), suggesting the potential for bacteria to begin the process of breaking down the fixed carbon compounds secreted by the photobiont for easier metabolism by the mycobiont. This first look at the group of functional genes expressed at the level of transcription provides initial insights into the symbiotic network of interacting genes within the lichen microbiome.

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.

Lichens are symbioses between a fungus and a photosynthesizing partner such as a green alga or a cyanobacterium. Unlike mycorrhizal or rhizobial symbioses, the lichen symbiosis is not well understood either morphologically or molecularly. The lichen symbiosis has been somewhat neglected for several reasons. Lichens grow very slowly in nature (less than 1 cm a year), it is difficult to grow the fungus and the alga separately and, moreover, it remains difficult to resynthesize the mature symbiosis in the laboratory. It is not yet possible to delete genes, nor has any transformation method been established to introduce genes into the genomes of either the fungus or the alga. However, the lack of genetic tools for these organisms has been partially compensated for by the sequencing of the genomes of the lichenizing fungus Cladonia grayi and its green algal partner Asterochloris sp. This work uses the model lichen system Cladonia grayi and the associated genomes to explore one evolutionary and one developmental question concerning the lichen symbiosis.

Chapter One uses data from the genomes to assess whether there was evidence of horizontal gene transfer between the lichen symbionts in the evolution of this very intimate association; that is, whether genes of algal origin could be found in the fungal genome or vise versa. An initial homology search of the two genomes demonstrated that the fungus had, in addition to ammonium transporter/ammonia permease genes that were clearly fungal in origin, ammonium transporter/ammonia permease genes which appeared to be of plant origin. Using cultures of various lichenizing fungi, plant-like ammonium transporter/ammonia permease genes were identified by degenerate PCR in ten additional species of lichen in three classes of lichenizing fungi including the Lecanoromycetes, the Eurotiomycetes, and the Dothidiomycetes. Using the sequences of these transporter genes as well as data from publically available genome sequences of diverse organisms, I constructed a phylogy of 513 ammonium transporter/ammonia permease sequences from 191 genomes representing all main lineages of life to infer the evolutionary history of this family of proteins. In this phylogeny I detected several horizontal gene transfer events, including the aforementioned one which was demonstrated to be not a transfer from plants to fungi or vise versa, but a gene gain from a group of phylognetically unrelated hyperthermophilic chemoautolithotrophic prokaryotes during the early evolution of land plants (Embryophyta), and an independent gain of this same gene in the filamentous ascomycetes (Pezizomycotina), which was subsequently lost in most lineages but retained in even distantly related lichenized fungi. Also demonstrated was the loss of the native fungal ammonium transporter and the subsequent replacement of this gene with a bacterial ammonium transporter during the early evolution of the fungi. Several additional recent horizontal gene transfers into lineages of eukaryotes were demonstrated as well. The phylogenetic analysis suggests that what has heretofore been conceived of as a protein family with two clades (AMT/MEP and Rh) is instead a protein family with three clades (AMT, MEP, and Rh). I show that the AMT/MEP/Rh family illustrates two contrasting modes of gene transmission: AMT family as defined here exhibits standard parent-to-offspring inheritance, whereas the MEP family as defined here is characterized by several ancient independent horizontal gene transfers (HGTs) into eukaryotes. The clades as depicted in this phylogenetic study appear to correspond to functionally different groups, with ammonium transporters and ammonia permeases forming two distinct and possibly monophyletic groups.

In Chapter Two I address a follow-up question: in key lichenizing lineages for which ammonium transporter/ammonia permease (AMTP) genes were not found in Chapter One, were the genes lost? The only definitive infomation which can demonstrate absence of a gene from a genome is a full genome sequence. To this end, the genomes of eight additional lichenizing fungi in the key clades including the Caliciales (sensu Gaya 2011), the Peltigerales, the Ostropomycetidae, the Acarosporomycetidae, the Verrucariales, the Arthoniomycetidae and the Lichinales were sequenced using the Ilumina HiSeq technology and assembled with the short reads assembly software Velvet. These genomes were searched for ammonium transporter/ammonia permease sequences as well as 20 test genes to assess the completeness of each assembly. The genes recovered were included in a refined phylogenetic analysis. The hypothesis that lichens symbiotic with a nitrogen-fixing cyanobacteria as a primary photobiont or living in high nitrogen environments lose the plant-like ammonium transporters was upheld, but did not account for additional losses of ammonium transporters/ammonia permeases in the Acarosporomyetidae and Arthoniomycetes. In addition, the four AMTP genes from Cladonia grayi were shown to be functional by expression of the lichen genes in a strain of Saccharomyces cerevisiae in which all three native ammonium transporters were deleted, and assaying for growth on limiting ammonia as a sole nitrogen source.

In Chapter Three I use genome data to address a developmental aspect of the lichen symbiosis. The finding that DNA in three genera of lichenizing fungi is methylated in symbiotic tissues and not methylated in aposymbiotic tissues or in the free-living fungus (Armaleo & Miao 1999a) suggested that epigenetic silencing may play a key role in the development of the symbiosis. Epigenetic silencing involves several steps that are conserved in many eukaryotes, including methylation of histone H3 at lysine 9 (H3K9) in nucleosomes within the silenced region, subsequent binding of heterochromatin-binding protein (HP1) over the region, and the recruitment of DNA methyltransferases to methylate the DNA, all of which causes the underlying chromatin to adopt a closed conformation, inhibiting the transcriptional machinery from binding. In this chapter I both identify the genes encoding the silencing machinery and determine the targets of the silencing machinery. I use degenerate PCR and genome sequencing to identify the genes encoding the H3K9 histone methyltransferase, the heterochromatin binding protein, and the DNA methyltransferases. I use whole genome bisulfite sequencing of DNA from the symbiotic structures of Cladonia grayi including podetia, squamules and soredia as well as DNA from cultures of the free-living fungus and free-living alga to determine which regions of the genome are methylated in the symbiotic and aposymbiotic states. In particular I examine regions of the genomes which appear to be differentially methylated in the symbiotic versus the aposymbiotic state. I show that DNA methylation is uncommon in the genome of the fungus in the symbiotic and aposymbiotic states, and that the genome of the alga is methylated in the symbiotic and aposymbiotic states.

Symbiosis is a ubiquitous phenomenon across the tree of life. In the fungal subphylum Pezizomycotina, symbiotic interactions with land plants, green algae, and cyanobacteria are ecologically diverse and have led to the proliferation of species. I address three main questions connected to Lecanoromycetes and Eurotiomycetes, sister classes that have taken dramatically different trajectories: Lecanoromycetes are almost all lichenized fungi, while Eurotiomycetes includes lifestyles as diverse as animal pathogens, plant mutualists, and rock-inhabiting extremophiles. (1) What is the species- level biodiversity of lichen-associated Trebouxia, and how do these species vary in their ecological interactions and environmental niches? (2) How do lichenized fungi and algae contribute to the biodiversity of southern Africa, and do they follow regional biogeographic patterns associated with vascular plants? (3) How are different trophic modes and lifestyles distributed in the phylogeny of Eurotiomycetes, and how do these lifestyles differ (or not) at a genomic level? In the first research chapter, I show that the distributions of Lecanoraceae fungi and their Trebouxia algal partners in Bolivia are structured by elevation. Maximum species turnover in the fungi and algae occur at different elevations, suggesting that the partners respond differently to change along the elevation gradient. I also contribute to the development of a phylogenetic classification for Trebouxia, recognizing several novel, putative species from Bolivia based on internal transcribed spacer (ITS) and rbcL data. In the second research chapter, I continue to develop the classification for Trebouxia using data from South Africa and Namibia. Ninenovel, putative species are proposed, and several putative species recognized in iv previous work are reassessed or rejected. I show that there is no evidence for the Greater Cape Floristic Region being a unique source for endemic Trebouxia species; conversely, there is evidence for extensive inter-biome sharing. In the third research chapter, I explore lichen species described by Nylander from South Africa in the 1860s. I show that the collector was likely Olivia Wolfenden Armstrong, that the collection locality is the Karkloof area, and that a species later described from the same region, Bacidia triphragmia (Stizenb.) Zahlbr., is a synonym of Nylander’s species Bacidia inconveniens (Nyl.) Zahlbr. The fourth research chapter continues to address the systematics of lichen- forming fungi in South Africa, focusing on the hyaline-spored species of Graphidaceae tribe Graphideae. Most South African collections from this group belong to pantropical species; an exception is the new species Allographa oldayana I. Medeiros, a putative endemic to forest fragments in eastern South Africa. I make the new combination Mangoldia bylii (Vain.) I. Medeiros and show that it is an earlier synonym for Mangoldia atronitens (A. W. Archer) Lücking et al. In the final research chapter, I obtain genomic and multilocus sequence data for endophytic, endolichenic, lichenicolous, lichenized, and bryophilous Eurotiomycetes and put these trophic modes in a phylogenetic context. There is no evidence for hybridization in the phylogeny of Phaeomoniellales, suggesting that difficulties inferring phylogenetic relationships among genera are likely due to incomplete lineage sorting. The new combination Knufia muscicola (Racov.) I. Medeiros & Greiff and the new genus Pseudoepibryon I. Medeiros & Greiff are proposed to partially resolve the polyphyly of Epibryon. Comparison of CAZyme and protease diversity does not show differences between endolichenic and lichenicolous Chaetothyriales.

Lichen-forming fungi employ a successful mode of nutrition as symbiotic partners with green algae and/or cyanobacteria (the photobiont). Nearly one fifth of all known fungi are obligate lichen formers, yet we know little of how they find compatible partners and establish long-lived symbiotic relationships. The combined growth of these symbionts forms a body (thallus) with emergent properties unlike either of the symbionts individually grown. Based on other well-studied eukaryotic systems, the development of a lichen thallus must rely upon the successful identification and collaboration of these two very different organisms. Identifying the molecular basis of microbe recognition and interactions remains one of the greatest challenges in studying symbiotic systems.

In this thesis, I determine the stage in which to begin looking for lichen symbiosis specific genes, and then examine mycobiont and photobiont genes that, when compared to the aposymbiotic state, are upregulated in the symbiotic state. Using the symbiosis between the mycobiont Cladonia grayi and the photobiont Asterochloris sp., as well as scanning electron microscopy observations of the earliest stages of contact between C. grayi and Asterochloris sp., I determined that the mycobiont undergoes a specific change in phenotypic growth in response to Asterochloris sp. This change is particular to the lichen symbiosis, and is not observed with algal shaped inanimate objects or algae other than Asterochloris. I then used this phenotypically defined stage that is exclusive to lichen symbiosis to begin studying the the genetic and molecular mechanisms underlying the development of a stratified lichen thallus. Using suppression subtractive hybridization to determine differential gene expression, fungal and algal libraries were made of upregulated genes in the first 2 stages of lichen symbiosis. The symbiotic expression levels of select genes were then verified using quantitative PCR. Lastly, a candidate gene for involvement in lichen symbiosis was transformed into Saccharomyces cerevisiae to test for protein function.

Further results of this study show that the fungal protein products of genes upregulated in lichen symbiosis show significant matches to proteins putatively involved in fungal self and non-self recognition, lipid metabolism, negative regulation of glucose repressible genes, an oxidoreductase, a dioxygenase, and a conserved hypothetical protein. Algal genes that are upregulated in lichen symbiosis include a chitinase-like protein, an amino acid metabolism protein, a dynein related protein, and a protein arginine methyltransferase. Furthermore, genes that are expressed in the early stages of lichen symbiosis are common varying metabolic pathways. Furthermore stages 1 and 2 of development are marked not by a drastic change in transcriptional products, but instead by an overall change in genes that are already expressed. Finally, the Cladonia~grayi Lip3was cloned in its entirety from genomic DNA and cDNA, was predicted to be secreted using signal peptide prediction software, and shown to be a functioning secreted extracellular lipase in yeast.

I conclude that many genes are involved in the interactions of symbionts and the development of a stratified lichen thallus, and that many more genes remain to be discovered. Furthermore, the possibility that genes exist in either symbiont that are specific to lichen symbiosis remains, and that their discovery awaits the creation of better genomic tools for \textit{Cladonia~grayi} and Asterochloris.