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<p>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 <italic>Cladonia
grayi</italic> and its green algal partner <italic>Asterochloris</italic> sp. This
work uses the model lichen system <italic>Cladonia grayi</italic> and the associated
genomes to explore one evolutionary and one developmental question concerning the
lichen symbiosis.</p><p>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.</p><p>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 <italic>Cladonia grayi</italic> were shown to
be functional by expression of the lichen genes in a strain of <italic>Saccharomyces
cerevisiae</italic> in which all three native ammonium transporters were deleted,
and assaying for growth on limiting ammonia as a sole nitrogen source. </p><p>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 <italic>Cladonia grayi</italic> 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.</p>
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