BACKGROUND
the nutrient-sensing target of rapamycin  pathway is highly conserved among eukaryotes and governs several essential cellular processes including protein synthesis, ribosome biogenesis, autophagy, and cytoskeletal organization  <cit> .
in the fungal kingdom, the tor pathway has been best studied in the budding yeast saccharomyces cerevisiae  <cit> , the fission yeast schizosaccharomyces pombe  <cit> , and the human pathogen candida albicans  <cit> .
however, little is known about tor in basal fungal lineages, including the zygomycota and chytridiomycota.
in s. cerevisiae and s. pombe, two tor paralogs form distinct complexes known as tor complex  <dig>  and tor complex  <dig>   <cit> , while in most other species, including humans, a single tor protein can populate both complexes  <cit> .
interestingly, s. cerevisiae tor <dig> can complement the loss of tor <dig>  but tor <dig> cannot complement the loss of tor <dig>  <cit> .
two tor paralogs have also been identified in a metazoan, the silkworm bombyx mori  <cit>  and three tor paralogs were identified in the trypanosomatid parasites leishmania major  <cit>  and the related species trypanosoma brucei  <cit> , the first reported tor triumvirates.
the sctorc <dig> is sensitive to rapamycin and controls protein synthesis, mrna synthesis and degradation, ribosome biogenesis, and autophagy.
torc <dig> is insensitive to rapamycin and is involved in the control of actin polarization and cell wall integrity  <cit> .
torc <dig> consists of tor <dig> or tor <dig>  kog <dig>  <cit> , tco <dig>  and lst <dig>  <cit> , while torc <dig> contains tor <dig>  lst <dig>  avo <dig>  avo <dig>  and avo <dig>  <cit>  .
recently, the ego-gtpase complex and its orthologs were shown to convey amino acid signals for torc <dig> activation in yeast, insects, and mammals  <cit> .
in s. cerevisiae, immediate effectors of torc <dig> include the pp2a-like phosphatase sit <dig>  <cit>  and the agc kinase sch <dig>  <cit> .
in s. pombe, tor participates in other cellular functions including nutrient signaling  <cit> , cell growth and differentiation  <cit> , mitotic commitment  <cit> , and sexual development  <cit>  .
the structure of the tor proteins is remarkably conserved  and features several domains for protein-protein interactions including n-terminal heat  repeats  <cit> , a fat  domain  <cit> , and an frb   <cit>  domain.
the kinase and the fatc   <cit>  domains are present in the c-terminal region.
the tor inhibitor rapamycin blocks cell proliferation and is currently used as an immunosuppressive drug for organ and tissue transplant recipients and a chemotherapy agent against a variety of solid cancers  <cit> .
rapamycin binds to the prolyl isomerase fkbp <dig> to form a protein-drug complex that then interacts with the tor frb domain in a ternary complex  <cit> .
s. cerevisiae cells treated with rapamycin display phenotypes associated with nutrient depletion including g <dig> cell cycle arrest, cellular volume expansion, protein synthesis inhibition, glycogen accumulation, and autophagy  <cit> .
genetic analysis in s. cerevisiae characterizing rapamycin-resistant mutants led to the identification of fkbp <dig> as the intracellular receptor for rapamycin and defined tor <dig> and tor <dig> as the targets of the fkbp12-rapamycin complex  <cit> .
subsequent studies resulted in the identification and characterization of the tor <dig> and tor <dig> gene products  <cit>  as well as the elucidation of the tor signaling cascade  <cit> .
these pioneering studies aided in the identification of the mammalian tor ortholog and characterization of this highly conserved signaling cascade  <cit> .
remarkably, expression of the human fkbp <dig> ortholog in yeast fpr <dig>  deletion mutants complements to restore rapamycin sensitivity, and hybrid tor proteins consisting of the yeast n-terminal domain fused to the mammalian tor kinase domain are also functional in yeast  <cit> .
thus, the tor pathway has been functionally and structurally conserved from yeasts to humans over the billion years of evolution separating the two species from their last common ancestor.
we now know that the fungal and metazoan kingdoms are both within the opisthokont lineage of eukaryotes and are thus more closely related to each other than the vast majority of eukaryotic organisms  <cit> .
moreover, these two highly successful kingdoms shared a last common ancestor as recently as one billion years ago, much more recently than most eukaryotes.
recent initiatives, particularly the unicorn project, have facilitated the sequencing of several opisthokonts related to the last common ancestor of the metazoans and fungi, providing an interesting window into the evolution of both fungi and animals  <cit> .
thus, studies on the evolutionary trajectory of the fungal kingdom in general, and of the tor signaling cascade in particular, promise to reveal insights about how orthologous pathways function in the more complex milieu of multicellular metazoan organisms.
in this study, we have capitalized upon the wealth of available genomic information by annotating the tor pathway in several fungal organisms in which this pathway has not been described.
our study included selected species in the major groups of the fungal kingdom with genome sequences available: the basidiomycete pleurotus ostreatus; the mucorales zygomycetes mucor circinelloides, rhizopus oryzae, and phycomyces blakesleeanus; the chytridiomycetes spizellomyces punctatus and batrachochytrium dendrobatidis; the microsporidian species encephalitozoon cuniculi, enterocytozoon bieneusi, and nosema ceranae; and the related non-fungal opisthokonts capsaspora owczarzaki, salpingoeca rosetta, and monosiga brevicollis.
whereas the tor pathway is conserved throughout the eukaryotes, strikingly, microsporidian species with their highly reduced and compacted genomes lack the entire tor pathway.
we have also investigated gene and genome duplication events that resulted in two tor homologs in s. cerevisiae, s. pombe, p. ostreatus, and b. dendrobatidis, and the loss of a second tor homolog following a whole genome duplication event in r. oryzae.
RESULTS
conservation of the tor signaling pathway molecular components
the tor pathway is well conserved among nearly all eukaryotic species examined to date.
tor is essential for life and is the target of the potent drug rapamycin in fungi, humans, and other eukaryotic organisms  <cit> .
tor originated early during the eukaryotic radiation, as it is present in the basal eukaryotes giardia lamblia  <cit> , l. major  <cit>  and t. brucei  <cit> , and also in plants  <cit> .
here we focused on the tor pathway in the fungal kingdom and other representative unicellular opisthokont species outside of the fungi.
several genes encoding tor complex components, upstream regulators, and downstream effectors were identified in all major groups throughout the fungal kingdom .
because we used blastp reciprocal best hits  to identify orthologs, in some cases the absence of an ortholog in our results may represent only a failure to detect it with this method.
further, functional homologs that are not sufficiently similar enough in sequence to be identified may be present.
overall, a high degree of pathway conservation is observed in the tor signaling cascade throughout the fungal kingdom, with the exception of microsporidia in which all tor pathway components are absent.
in addition, several pathway components are conserved in the related unicellular opisthokonts m. brevicollis, s. rosetta, and c. owczarzaki .
the presence or absence of tor pathway components is indicated by + or -, respectively.
abbreviations: sc = s. cerevisiae, sp = s. pombe, po = p. ostreatus, mc = m. circinelloides, ro = r. oryzae, pb = p. blakesleeanus, bd = b. dendrobatidis, spu = s. punctatus.
the presence of absence of tor pathway components is indicated by + or -, respectively.
abbreviations: mb = m. brevicollis, sr = s. rosetta, co = c. owczarzaki.
in s. cerevisiae, tor <dig> and tor <dig> interact with several proteins to form torc <dig> and torc <dig>  torc <dig> contains tor <dig> or tor <dig>  kog <dig>  tco <dig>  and lst <dig>  kog <dig> functions as a substrate-recruiting subunit in mammalian torc <dig>   <cit> .
a gene encoding a putative kog <dig> homolog was identified in all species included in this study, with the exception of the microsporidia and s. rosetta .
mutation of tco <dig> results in hypersensitivity to rapamycin and affects cellular integrity in s. cerevisiae  <cit> .
tco <dig> is only found in s. cerevisiae and could have resulted from a specialization of the pathway exclusive to saccharomyces species .
however, while a blastp search did not identify tco <dig> homologs in other species, s. pombe has been reported to contain a functional homolog   <cit> , and this suggests that functional homologs may exist in other fungal species as well.
however, no homologs were identified when using s. pombe tco <dig> as a query sequence.
lst <dig> binds to the tor kinase domain in s. cerevisiae to stimulate catalytic activity  <cit>  and a putative lst <dig> homolog is conserved in most species analyzed except microsporidia and s. rosetta .
additional blastp searches identified a single tor homolog in the following species: lachancea thermotolerans cbs <dig> , pichia pastoris gs <dig> , pichia stipitis cbs <dig> , debaryomyces hansenii cbs <dig> , yarrowia lipolytica clib <dig> , podospora anserina strain s mat+ , chaetomium globosum cbs <dig>  , magnaporthe oryzae 70- <dig> , gibberella zeae ph- <dig> , aspergillus fumigatus af <dig> , aspergillus flavus nrrl <dig> , neosartorya fischeri nrrl <dig> , aspergillus terreus nih <dig> , aspergillus nidulans fgsca <dig> , aspergillus clavatus nrrl <dig> , aspergillus oryzae rib <dig> , and aspergillus niger cbs <dig>  .
these ascomycetes were not included in further studies.
in yeast and mammals, torc <dig> controls spatial aspects of growth.
torc <dig> includes tor <dig>  avo <dig>  avo <dig>  avo <dig>  and bit <dig>  <cit> .
avo <dig> plays an essential role in actin cytoskeleton polarization  <cit>  and is conserved throughout the organisms that were the focus of this study  except in s. rosetta and c. owczarzaki .
however, due to high divergence of the avo <dig> sequence amongst the known homologs, it is possible that the s. rosetta and c. owczarzaki homologs cannot be identified using the blastp algorithm.
avo <dig> is a nonessential substrate adaptor for torc <dig>  <cit>  and potential homologs were identified in most species studied, with the exceptions of s. pombe, p. ostreatus, the three microsporidian species , and m. brevicollis .
in s. cerevisiae, avo <dig>  controls cytoskeletal dynamics  <cit> , and homologs are conserved throughout the species examined .
bit <dig>  a nonessential protein that associates with torc <dig>  <cit> , was only identified in s. cerevisiae .
upstream regulators of tor include tsc <dig> and tsc <dig>  and the gtpase rheb .
in s. pombe and other eukaryotes, tsc <dig> and tsc <dig> form a gtpase-activating complex that negatively regulates the action of rheb to activate torc <dig>  <cit> .
tsc <dig> and tsc <dig> putative homologs were identified in most species in the study, with the exception of s. cerevisiae ; however, tsc <dig> homologs were not identified in b. dendrobatidis, s. rosetta, or c. owczarzaki .
in mammals and insects, four rag gtpases  bind raptor  to mediate torc <dig> signaling in response to amino acids  <cit> .
raga and ragb are orthologs of s. cerevisiae gtr <dig> whereas ragc and ragd are orthologs of s. cerevisiae gtr <dig>  <cit> .
gtr <dig> and gtr <dig> form a complex along with ego <dig> and ego <dig> known as egoc/gse  <cit> , which is regulated by the gtp exchange factor vam <dig>  <cit> .
interestingly we found presumptive gtr <dig> and gtr <dig> orthologs in species in this study with the exception of microsporidia , and gtr <dig> was not identified in m. brevicollis .
however, ego <dig> and ego <dig>  which anchor gtr <dig> and gtr <dig> to endosomal and vacuolar membranes, are unique to s. cerevisiae and were not identified in the other species .
this suggests that the specific ego complex architecture might be restricted to saccharomyces, though it is possible that functional homologs may be present in other species.
in fungi, several downstream effectors are targets of tor signaling.
the pp2a phosphatase sit <dig> and its regulatory subunit tap <dig> regulate the expression of several torc1-controlled genes and gcn2-regulated translation  <cit> .
a sit <dig> homolog was identified in r. oryzae , though a homolog of the regulatory subunit tap <dig> was not .
in s. rosetta, a sit <dig> homolog was not identified, though tap <dig> was .
both of these situations could indicate rewiring of the pathway, or alternatively these findings could be a result of insufficient similarity to detect with a rbh search method.
sch <dig>  a member of the agc kinase family and the functional homolog of the p <dig> s <dig> kinase, is a direct substrate of s. cerevisiae torc <dig> involved in ribosome biogenesis  <cit> .
sch <dig> was not identified in rbh blastp searches with s. cerevisiae sch <dig> in m. circinelloides, p. blakesleeanus, the microsporidia , or s. rosetta , but was identified in all other species.
putative homologs of ypk <dig> kinase, a regulator of ceramide synthesis controlled by torc <dig> signaling  <cit> , were only identified in s. cerevisiae, s. pombe, , m. brevicollis, s. rosetta, and c. owczarzaki .
notably, the failure to detect many homologs in m. brevicollis, s. rosetta, and c. owczarzaki could be due to their evolutionary distance from the other organisms in the study and their gene divergence.
evolutionary conservation of tor kinase protein structure
the tor kinases are phosphatidylinositol kinase-related kinases   <cit>  and aside from their known protein kinase function also serve as evolutionary constrained scaffolds with several protein-protein interaction domains that mediate multi-protein complex formation  <cit> .
tor protein architecture was annotated using smart  <cit>  and we found that it has been maintained throughout the species of this study .
n-terminal heat repeats are present in varying numbers and serve as scaffolding structures for protein-protein interactions  <cit> .
similarly, the fat/toxic domain and the accompanying c-terminal fatc domain also participate in protein scaffolding and are present and conserved in all species subject to analysis.
over-expression of the fat domain has a dominant negative effect on cell growth in yeast and this effect can be suppressed by over-expression of phospholipase c  <cit> .
the kinase domain has similarities to both lipid and protein kinases, but it is a bona fide protein kinase.
the frb  domain is a highly conserved  <dig> amino acid region of tor with several residues that are known to be required for binding the fkbp12-rapamycin drug complex  <cit> .
in addition, the frb domain contains several residues necessary for both phosphatidic acid binding and rapamycin interaction  <cit> .
in mammals, phosphatidic acid binding to the frb region may promote the assembly of mtor complexes.
it has been proposed that the potent effect of the tor inhibitor rapamycin may be due to direct competition for overlapping phosphatidic acid binding sites within the frb domain  <cit> .
mutation of the conserved amino acid residues s <dig>  w <dig>  or f <dig> in the frb domain confers rapamycin resistance in s. cerevisiae  <cit> .
strikingly, the corresponding amino acid residues are conserved in all species examined .
in mtor, phosphatidic acid binding is disrupted by mutations of l <dig>  f <dig>  and y <dig>  <cit> , corresponding to s. cerevisiae tor <dig> l <dig>  f <dig>  and y <dig>  respectively, all of which are conserved in the studied fungal species .
duplication of tor in the fungal kingdom
approximately  <dig> million years ago, the s. cerevisiae ancestor underwent a whole genome duplication  event, and species that descend from this evolutionary event retained duplicated subsets of genes.
s. cerevisiae retained approximately 8% of duplicated genes  <cit> , while other fungi in this lineage have maintained varying numbers of gene duplicates, such as candida glabrata in which a smaller number  of these pairs are maintained  <cit> .
interestingly, several of these species, including s. cerevisiae, s. paradoxus, s. mikatae, s. kudriavzevii, s. bayanus, s. castellii, and c. glabrata have retained two tor paralogs, whereas species outside of the wgd clade, including kluyveromyces lactis and ashbya gossypii, have only a single tor gene.
the fates of duplicated gene paralogs can be explained through the duplication, degeneration, and complementation  model of gene duplication  <cit> .
in species with a single tor, this protein populates both torc <dig> and torc <dig>  each of which has distinct functions.
following duplication, each paralog likely sub-functionalized to carry out some, but not all, of its previous functions so that between the two paralogs, each process is performed.
in s. cerevisiae, for example, tor <dig> exclusively functions within torc <dig>  while tor <dig> preferentially populates torc <dig> but can also function in torc <dig>  s. pombe has two tor paralogs as a result of an independent segmental gene duplication event, and each paralog has distinct roles not necessarily equivalent to those of s. cerevisiae tor <dig> and tor <dig>  sptor <dig> is not essential, whereas sptor <dig> is essential for growth  <cit> .
we hypothesize that similar models could apply in species encoding two tor homologs resulting from independent gene duplication events such as p. ostreatus and b. dendrobatidis.
based on their level of identity with s. cerevisiae tor homologs, the b. dendrobatidis genes bdeg_ <dig> and bdeg_ <dig> have been designated tor <dig> and tor <dig>  respectively.
however, these gene names are not necessarily based on functional similarity with sctor <dig> and sctor <dig> but rather indicate only that there are two tor homologs in b. dendrobatidis.
a similar naming challenge is evident in s. pombe, where the functions of sptor <dig> and sptor <dig> are not equivalent with those of sctor <dig> and sctor <dig>
while several species, such as those in the saccharomyces clade, have two tor paralogs resulting from ancient wgd events, other species have acquired a second paralog through independent segmental gene duplication events, including b. dendrobatidis and several fission yeast species closely related to s. pombe .
segmental gene duplications have played a major role in the evolution of species and can result in the duplication of single genes or large blocks of genes  <cit> .
in s. cerevisiae, the duplication blocks with tor include three paralogous gene pairs that have been retained from the ancestral wgd event: ptk1/ptk <dig>  tor1/tor <dig>  and mnn4/yjr061w  <cit>  .
further, c. glabrata, an ascomycete closely related to s. cerevisiae that diverged following the wgd, has also maintained a second tor paralog and synteny is apparent in the region flanking the tor <dig> and tor <dig> genes, though one must extend the analysis to > <dig> kb from the tor genes to detect it .
however, because c. glabrata and s. cerevisiae have retained different subsets of genes from the ancestral wgd, the duplicated genes surrounding tor in these species are not homologs of one another.
further, c. glabrata has maintained fewer of the synteny blocks found in s. cerevisiae, and the block containing the tor genes  is not conserved in c. glabrata  <cit> .
in contrast, genomic regions surrounding the tor paralogs in s. pombe  and b. dendrobatidis  are not syntenically conserved.
that is, the duplicated genes are not flanked by other duplicated gene pairs, and the two tor paralogs likely resulted from independent segmental gene duplication events.
further, there are no indications of wgds in these species.
in the schizosaccharomycotina, the tor gene duplication occurred prior to speciation as evidenced by the fact that s. pombe, s. octosporus, s. japonicus, and s. cryophilus have each retained a second tor paralog; the tor orthologs across these four species are more closely related to each other than to those from other species .
no syntenic conservation between the genomic regions containing tor paralogs was detected in any species of the schizosaccharomyces group, supporting the occurrence of an independent segmental gene duplication in their common ancestor .
the taphrinomycotina is a monophyletic taxon of the ascomycota containing the schizosaccharomycetes, taphrinomycetes, neolectomycetes, and pneumocystidomycetes.
genomes are only available for species within the schizosaccharomycetes  and the taphrinomycetes .
while the studied schizosaccharomyces spp.
contain two tor homologs, a single homolog was identified in p. carinii with tblastx using the highly conserved s. cerevisiae frb domain of tor <dig> based on sequence data produced by the pneumocystis genome project  that was obtained from http://pgp.cchmc.org at the time of publication.
based on the phylogenetic placement of these two groups  <cit>  and the available genomic information, we hypothesize that the segmental tor duplication is unique to the schizosaccharomyces spp.
and occurred following their divergence from the common ancestor of the taphrinomycota, as p. carinii, the other species within this taxon with available genome data, appears to contain a single tor homolog.
interestingly, the zygomycete r. oryzae underwent a whole genome duplication event and despite retaining 12% of the resulting duplicated genes  <cit>  compared to 8% retained genes in a separate wgd event in s. cerevisiae  <cit> , two tor paralogs have not been retained; only a single tor homolog was identified.
currently, the zygomycota is the only group studied without an identified species containing two tor homologs; however, there are not enough available genomes to make a definitive conclusion in this group.
other species with independent tor duplications likely remain to be identified in all of the major fungal lineages, and as more genome sequences become available this will be addressed.
during this study, we identified three additional species that possibly have two tor paralogs: m. brevicollis, thecamonas trahens, and allomyces macrogynus, species that are included in the origins of multicellularity project  <cit> .
while analysis is limited because of the state of the m. brevicollis  genome, it appears that an additional bonafide tor protein is encoded with the conserved tor protein architecture.
thecamonas trahens is a unicellular apusomonad that also contains two tor paralogs.
interestingly, a. macrogynus is a fungus that may contain a second paralog; however, it appears that the frb domain overlaps with the kinase domain.
this may represent a case in which tor was duplicated but is in the process of losing one of the copies.
these cases are a springboard for future studies involving the duplication of tor within and beyond the fungal kingdom.
loss of the tor signaling pathway in microsporidia
while components of the tor signaling cascade are conserved in eukaryotes from yeasts to humans, the microsporidia, a group of highly specialized obligate intracellular pathogens completely lack all of the pathway components investigated in this study.
e. cuniculi has previously been noted to lack the tor kinase  <cit> , and we found that all pathway components including tor were missing not only in e. cuniculi but also in its microsporidian relatives e. bieneusi and n. ceranae .
microsporidian genomes are highly compacted and show a marked reduced gene content.
the genome of the human pathogen e. cuniculi has  <dig> genes distributed throughout  <dig> chromosomes in a  <dig>  mb genome  <cit> , less than half the number of protein-coding genes found in escherichia coli.
for comparison, the genome of the human pathogen e. bieneusi genome is ~ <dig> mb with  <dig>  genes  <cit>  and the honeybee pathogen n. ceranae genome is ~ <dig>  mb with  <dig>  predicted genes  <cit> .
genome reduction has been well studied in intracellular bacteria: during the specialization process from a free-living to intracellular lifestyle, massive gene loss can occur  <cit> .
we hypothesize that as a consequence of their specialization to an obligate intracellular lifestyle, the need for nutrient sensing was relaxed and these obligate intracellular microsporidia acquired nutrients from within the host cytoplasm.
thus, they no longer required the tor pathway, which is dedicated to sensing changes in the nutrient composite of the environmental milieu.
however, e. cuniculi has retained some of the genes encoding signaling pathways involved in nutrient sensing in other species, including homologs of s. cerevisiae ras <dig> and ras <dig> .
interestingly, p. carinii is an obligate pathogen with a reduced genome estimated to be ~ <dig> mb  <cit> , which unlike microsporidia is not obligately intracellular.
a single tor homolog was identified  using the s. cerevisiae tor <dig> frb domain in tblastx, supporting our hypothesis that the microsporidia have streamlined their genomes as they adopted an obligate intracellular life cycle.
p. carinii likely requires tor because it must survive in a metabolically active form outside of host cells, undergoing cell division in the extracellular milieu of the infected host lung.
many microsporidian genomes have undergone extreme genome reduction and are among the eukaryotes with the smallest genomes.
in addition to fewer genes than other species , e. cuniculi has shorter genes, small intergenic distances, and very few introns  <cit> .
this species  lacks several genes that are necessary for biosynthetic pathways and the tricarboxylic acid cycle in free-living organisms  <cit> .
remarkably, e. cuniculi must acquire atp from its host through a series of atp transporters anciently acquired from rickettsia- or chlamydia-like bacteria  <cit> , and this is an example of how e. cuniculi, and possibly other microsporidia, can hijack mechanisms of other intracellular bacteria, fungi, or the host to survive in and adapt to their highly specialized intracellular lifestyle.
CONCLUSIONS
the tor pathway is highly conserved with some exceptions among fungi, including several pathogens.
similarly, the molecular structural organization of the tor kinases has been remarkably conserved as well as the presumed ability to bind the fkbp12-rapamycin complex.
this feature, combined with the essential nature of the rapamycin-sensitive torc <dig> pathway for cell growth, should enable the development of rapamycin-based strategies for antifungal therapies.
duplications of the tor protein occurred in most fungal groups examined, resulting from either independent segmental gene duplication events or a wgd event.
the maintenance of two tor homologs can be explained through the ddc model of gene duplication, in which the paralogs subfunctionalize and together support all of the functions of the pre-duplicated protein.
in addition, species outside of the fungal kingdom with two or more tor homologs include the metazoan b. mori, the apusomonad protozoan t. trahens, the trypanosome protozoans l. major and t. brucei, and possibly the choanoflagellate m. brevicollis.
this suggests that the tor gene has been independently duplicated multiple times since the last common ancestor of the opisthokonta and excavata lineages.
the tor pathway, as well as the tor protein itself, is highly conserved in eukaryotes, so the observation that three microsporidian species are missing the entire pathway is striking.
it would appear that the specialized obligate intracellular life cycle of microsporidia allows for the acquisition of nutrients from the host cell, obviating a requirement for this central nutrient-sensing pathway that is essential in all known cases of other eukaryotes for axenic growth.

 the nutrient-sensing target of rapamycin pathway is highly conserved among eukaryotes and governs several essential cellular processes including protein synthesis, ribosome biogenesis, autophagy, and cytoskeletal organization. in the fungal kingdom, the tor pathway has been best studied in the budding yeast saccharomyces cerevisiae, the fission yeast schizosaccharomyces pombe, and the human pathogen candida albicans. however, little is known about tor in basal fungal lineages, including the zygomycota and chytridiomycota. in s. cerevisiae and s. pombe, two tor paralogs form distinct complexes known as tor complex and tor complex, while in most other species, including humans, a single tor protein can populate both complexes. interestingly, s. cerevisiae tor can complement the loss of tor but tor cannot complement the loss of tor. two tor paralogs have also been identified in a metazoan, the silkworm bombyx mori and three tor paralogs were identified in the trypanosomatid parasites leishmania major and the related species trypanosoma brucei, the first reported tor triumvirates. the sctorc is sensitive to rapamycin and controls protein synthesis, mrna synthesis and degradation, ribosome biogenesis, and autophagy. torc is insensitive to rapamycin and is involved in the control of actin polarization and cell wall integrity. torc consists of tor or tor kog, tco and lst, while torc contains tor lst avo avo and avo. recently, the ego-gtpase complex and its orthologs were shown to convey amino acid signals for torc activation in yeast, insects, and mammals. in s. cerevisiae, immediate effectors of torc include the pp2a-like phosphatase sit and the agc kinase sch. in s. pombe, tor participates in other cellular functions including nutrient signaling, cell growth and differentiation, mitotic commitment, and sexual development. the structure of the tor proteins is remarkably conserved and features several domains for protein-protein interactions including n-terminal heat repeats, a fat domain, and an frb domain. the kinase and the fatc domains are present in the c-terminal region. the tor inhibitor rapamycin blocks cell proliferation and is currently used as an immunosuppressive drug for organ and tissue transplant recipients and a chemotherapy agent against a variety of solid cancers. rapamycin binds to the prolyl isomerase fkbp to form a protein-drug complex that then interacts with the tor frb domain in a ternary complex. s. cerevisiae cells treated with rapamycin display phenotypes associated with nutrient depletion including g cell cycle arrest, cellular volume expansion, protein synthesis inhibition, glycogen accumulation, and autophagy. genetic analysis in s. cerevisiae characterizing rapamycin-resistant mutants led to the identification of fkbp as the intracellular receptor for rapamycin and defined tor and tor as the targets of the fkbp12-rapamycin complex. subsequent studies resulted in the identification and characterization of the tor and tor gene products as well as the elucidation of the tor signaling cascade. these pioneering studies aided in the identification of the mammalian tor ortholog and characterization of this highly conserved signaling cascade. remarkably, expression of the human fkbp ortholog in yeast fpr deletion mutants complements to restore rapamycin sensitivity, and hybrid tor proteins consisting of the yeast n-terminal domain fused to the mammalian tor kinase domain are also functional in yeast. thus, the tor pathway has been functionally and structurally conserved from yeasts to humans over the billion years of evolution separating the two species from their last common ancestor. we now know that the fungal and metazoan kingdoms are both within the opisthokont lineage of eukaryotes and are thus more closely related to each other than the vast majority of eukaryotic organisms. moreover, these two highly successful kingdoms shared a last common ancestor as recently as one billion years ago, much more recently than most eukaryotes. recent initiatives, particularly the unicorn project, have facilitated the sequencing of several opisthokonts related to the last common ancestor of the metazoans and fungi, providing an interesting window into the evolution of both fungi and animals. thus, studies on the evolutionary trajectory of the fungal kingdom in general, and of the tor signaling cascade in particular, promise to reveal insights about how orthologous pathways function in the more complex milieu of multicellular metazoan organisms. in this study, we have capitalized upon the wealth of available genomic information by annotating the tor pathway in several fungal organisms in which this pathway has not been described. our study included selected species in the major groups of the fungal kingdom with genome sequences available: the basidiomycete pleurotus ostreatus; the mucorales zygomycetes mucor circinelloides, rhizopus oryzae, and phycomyces blakesleeanus; the chytridiomycetes spizellomyces punctatus and batrachochytrium dendrobatidis; the microsporidian species encephalitozoon cuniculi, enterocytozoon bieneusi, and nosema ceranae; and the related non-fungal opisthokonts capsaspora owczarzaki, salpingoeca rosetta, and monosiga brevicollis. whereas the tor pathway is conserved throughout the eukaryotes, strikingly, microsporidian species with their highly reduced and compacted genomes lack the entire tor pathway. we have also investigated gene and genome duplication events that resulted in two tor homologs in s. cerevisiae, s. pombe, p. ostreatus, and b. dendrobatidis, and the loss of a second tor homolog following a whole genome duplication event in r. oryzae.  conservation of the tor signaling pathway molecular components the tor pathway is well conserved among nearly all eukaryotic species examined to date. tor is essential for life and is the target of the potent drug rapamycin in fungi, humans, and other eukaryotic organisms. tor originated early during the eukaryotic radiation, as it is present in the basal eukaryotes giardia lamblia, l. major and t. brucei, and also in plants. here we focused on the tor pathway in the fungal kingdom and other representative unicellular opisthokont species outside of the fungi. several genes encoding tor complex components, upstream regulators, and downstream effectors were identified in all major groups throughout the fungal kingdom. because we used blastp reciprocal best hits to identify orthologs, in some cases the absence of an ortholog in our results may represent only a failure to detect it with this method. further, functional homologs that are not sufficiently similar enough in sequence to be identified may be present. overall, a high degree of pathway conservation is observed in the tor signaling cascade throughout the fungal kingdom, with the exception of microsporidia in which all tor pathway components are absent. in addition, several pathway components are conserved in the related unicellular opisthokonts m. brevicollis, s. rosetta, and c. owczarzaki. the presence or absence of tor pathway components is indicated by + or -, respectively. abbreviations: sc = s. cerevisiae, sp = s. pombe, po = p. ostreatus, mc = m. circinelloides, ro = r. oryzae, pb = p. blakesleeanus, bd = b. dendrobatidis, spu = s. punctatus. the presence of absence of tor pathway components is indicated by + or -, respectively. abbreviations: mb = m. brevicollis, sr = s. rosetta, co = c. owczarzaki. in s. cerevisiae, tor and tor interact with several proteins to form torc and torc torc contains tor or tor kog tco and lst kog functions as a substrate-recruiting subunit in mammalian torc. a gene encoding a putative kog homolog was identified in all species included in this study, with the exception of the microsporidia and s. rosetta. mutation of tco results in hypersensitivity to rapamycin and affects cellular integrity in s. cerevisiae. tco is only found in s. cerevisiae and could have resulted from a specialization of the pathway exclusive to saccharomyces species. however, while a blastp search did not identify tco homologs in other species, s. pombe has been reported to contain a functional homolog, and this suggests that functional homologs may exist in other fungal species as well. however, no homologs were identified when using s. pombe tco as a query sequence. lst binds to the tor kinase domain in s. cerevisiae to stimulate catalytic activity and a putative lst homolog is conserved in most species analyzed except microsporidia and s. rosetta. additional blastp searches identified a single tor homolog in the following species: lachancea thermotolerans cbs, pichia pastoris gs, pichia stipitis cbs, debaryomyces hansenii cbs, yarrowia lipolytica clib, podospora anserina strain s mat+, chaetomium globosum cbs, magnaporthe oryzae 70-, gibberella zeae ph-, aspergillus fumigatus af, aspergillus flavus nrrl, neosartorya fischeri nrrl, aspergillus terreus nih, aspergillus nidulans fgsca, aspergillus clavatus nrrl, aspergillus oryzae rib, and aspergillus niger cbs. these ascomycetes were not included in further studies. in yeast and mammals, torc controls spatial aspects of growth. torc includes tor avo avo avo and bit. avo plays an essential role in actin cytoskeleton polarization and is conserved throughout the organisms that were the focus of this study except in s. rosetta and c. owczarzaki. however, due to high divergence of the avo sequence amongst the known homologs, it is possible that the s. rosetta and c. owczarzaki homologs cannot be identified using the blastp algorithm. avo is a nonessential substrate adaptor for torc and potential homologs were identified in most species studied, with the exceptions of s. pombe, p. ostreatus, the three microsporidian species, and m. brevicollis. in s. cerevisiae, avo controls cytoskeletal dynamics, and homologs are conserved throughout the species examined. bit a nonessential protein that associates with torc, was only identified in s. cerevisiae. upstream regulators of tor include tsc and tsc and the gtpase rheb. in s. pombe and other eukaryotes, tsc and tsc form a gtpase-activating complex that negatively regulates the action of rheb to activate torc. tsc and tsc putative homologs were identified in most species in the study, with the exception of s. cerevisiae; however, tsc homologs were not identified in b. dendrobatidis, s. rosetta, or c. owczarzaki. in mammals and insects, four rag gtpases bind raptor to mediate torc signaling in response to amino acids. raga and ragb are orthologs of s. cerevisiae gtr whereas ragc and ragd are orthologs of s. cerevisiae gtr. gtr and gtr form a complex along with ego and ego known as egoc/gse, which is regulated by the gtp exchange factor vam. interestingly we found presumptive gtr and gtr orthologs in species in this study with the exception of microsporidia, and gtr was not identified in m. brevicollis. however, ego and ego which anchor gtr and gtr to endosomal and vacuolar membranes, are unique to s. cerevisiae and were not identified in the other species. this suggests that the specific ego complex architecture might be restricted to saccharomyces, though it is possible that functional homologs may be present in other species. in fungi, several downstream effectors are targets of tor signaling. the pp2a phosphatase sit and its regulatory subunit tap regulate the expression of several torc1-controlled genes and gcn2-regulated translation. a sit homolog was identified in r. oryzae, though a homolog of the regulatory subunit tap was not. in s. rosetta, a sit homolog was not identified, though tap was. both of these situations could indicate rewiring of the pathway, or alternatively these findings could be a result of insufficient similarity to detect with a rbh search method. sch a member of the agc kinase family and the functional homolog of the p s kinase, is a direct substrate of s. cerevisiae torc involved in ribosome biogenesis. sch was not identified in rbh blastp searches with s. cerevisiae sch in m. circinelloides, p. blakesleeanus, the microsporidia, or s. rosetta, but was identified in all other species. putative homologs of ypk kinase, a regulator of ceramide synthesis controlled by torc signaling, were only identified in s. cerevisiae, s. pombe,, m. brevicollis, s. rosetta, and c. owczarzaki. notably, the failure to detect many homologs in m. brevicollis, s. rosetta, and c. owczarzaki could be due to their evolutionary distance from the other organisms in the study and their gene divergence. evolutionary conservation of tor kinase protein structure the tor kinases are phosphatidylinositol kinase-related kinases and aside from their known protein kinase function also serve as evolutionary constrained scaffolds with several protein-protein interaction domains that mediate multi-protein complex formation. tor protein architecture was annotated using smart and we found that it has been maintained throughout the species of this study. n-terminal heat repeats are present in varying numbers and serve as scaffolding structures for protein-protein interactions. similarly, the fat/toxic domain and the accompanying c-terminal fatc domain also participate in protein scaffolding and are present and conserved in all species subject to analysis. over-expression of the fat domain has a dominant negative effect on cell growth in yeast and this effect can be suppressed by over-expression of phospholipase c. the kinase domain has similarities to both lipid and protein kinases, but it is a bona fide protein kinase. the frb domain is a highly conserved amino acid region of tor with several residues that are known to be required for binding the fkbp12-rapamycin drug complex. in addition, the frb domain contains several residues necessary for both phosphatidic acid binding and rapamycin interaction. in mammals, phosphatidic acid binding to the frb region may promote the assembly of mtor complexes. it has been proposed that the potent effect of the tor inhibitor rapamycin may be due to direct competition for overlapping phosphatidic acid binding sites within the frb domain. mutation of the conserved amino acid residues s w or f in the frb domain confers rapamycin resistance in s. cerevisiae. strikingly, the corresponding amino acid residues are conserved in all species examined. in mtor, phosphatidic acid binding is disrupted by mutations of l f and y, corresponding to s. cerevisiae tor l f and y respectively, all of which are conserved in the studied fungal species. duplication of tor in the fungal kingdom approximately million years ago, the s. cerevisiae ancestor underwent a whole genome duplication event, and species that descend from this evolutionary event retained duplicated subsets of genes. s. cerevisiae retained approximately 8% of duplicated genes, while other fungi in this lineage have maintained varying numbers of gene duplicates, such as candida glabrata in which a smaller number of these pairs are maintained. interestingly, several of these species, including s. cerevisiae, s. paradoxus, s. mikatae, s. kudriavzevii, s. bayanus, s. castellii, and c. glabrata have retained two tor paralogs, whereas species outside of the wgd clade, including kluyveromyces lactis and ashbya gossypii, have only a single tor gene. the fates of duplicated gene paralogs can be explained through the duplication, degeneration, and complementation model of gene duplication. in species with a single tor, this protein populates both torc and torc each of which has distinct functions. following duplication, each paralog likely sub-functionalized to carry out some, but not all, of its previous functions so that between the two paralogs, each process is performed. in s. cerevisiae, for example, tor exclusively functions within torc while tor preferentially populates torc but can also function in torc s. pombe has two tor paralogs as a result of an independent segmental gene duplication event, and each paralog has distinct roles not necessarily equivalent to those of s. cerevisiae tor and tor sptor is not essential, whereas sptor is essential for growth. we hypothesize that similar models could apply in species encoding two tor homologs resulting from independent gene duplication events such as p. ostreatus and b. dendrobatidis. based on their level of identity with s. cerevisiae tor homologs, the b. dendrobatidis genes bdeg_ and bdeg_ have been designated tor and tor respectively. however, these gene names are not necessarily based on functional similarity with sctor and sctor but rather indicate only that there are two tor homologs in b. dendrobatidis. a similar naming challenge is evident in s. pombe, where the functions of sptor and sptor are not equivalent with those of sctor and sctor while several species, such as those in the saccharomyces clade, have two tor paralogs resulting from ancient wgd events, other species have acquired a second paralog through independent segmental gene duplication events, including b. dendrobatidis and several fission yeast species closely related to s. pombe. segmental gene duplications have played a major role in the evolution of species and can result in the duplication of single genes or large blocks of genes. in s. cerevisiae, the duplication blocks with tor include three paralogous gene pairs that have been retained from the ancestral wgd event: ptk1/ptk tor1/tor and mnn4/yjr061w. further, c. glabrata, an ascomycete closely related to s. cerevisiae that diverged following the wgd, has also maintained a second tor paralog and synteny is apparent in the region flanking the tor and tor genes, though one must extend the analysis to > kb from the tor genes to detect it. however, because c. glabrata and s. cerevisiae have retained different subsets of genes from the ancestral wgd, the duplicated genes surrounding tor in these species are not homologs of one another. further, c. glabrata has maintained fewer of the synteny blocks found in s. cerevisiae, and the block containing the tor genes is not conserved in c. glabrata. in contrast, genomic regions surrounding the tor paralogs in s. pombe and b. dendrobatidis are not syntenically conserved. that is, the duplicated genes are not flanked by other duplicated gene pairs, and the two tor paralogs likely resulted from independent segmental gene duplication events. further, there are no indications of wgds in these species. in the schizosaccharomycotina, the tor gene duplication occurred prior to speciation as evidenced by the fact that s. pombe, s. octosporus, s. japonicus, and s. cryophilus have each retained a second tor paralog; the tor orthologs across these four species are more closely related to each other than to those from other species. no syntenic conservation between the genomic regions containing tor paralogs was detected in any species of the schizosaccharomyces group, supporting the occurrence of an independent segmental gene duplication in their common ancestor. the taphrinomycotina is a monophyletic taxon of the ascomycota containing the schizosaccharomycetes, taphrinomycetes, neolectomycetes, and pneumocystidomycetes. genomes are only available for species within the schizosaccharomycetes and the taphrinomycetes. while the studied schizosaccharomyces spp. contain two tor homologs, a single homolog was identified in p. carinii with tblastx using the highly conserved s. cerevisiae frb domain of tor based on sequence data produced by the pneumocystis genome project that was obtained from http://pgp.cchmc.org at the time of publication. based on the phylogenetic placement of these two groups and the available genomic information, we hypothesize that the segmental tor duplication is unique to the schizosaccharomyces spp. and occurred following their divergence from the common ancestor of the taphrinomycota, as p. carinii, the other species within this taxon with available genome data, appears to contain a single tor homolog. interestingly, the zygomycete r. oryzae underwent a whole genome duplication event and despite retaining 12% of the resulting duplicated genes compared to 8% retained genes in a separate wgd event in s. cerevisiae, two tor paralogs have not been retained; only a single tor homolog was identified. currently, the zygomycota is the only group studied without an identified species containing two tor homologs; however, there are not enough available genomes to make a definitive conclusion in this group. other species with independent tor duplications likely remain to be identified in all of the major fungal lineages, and as more genome sequences become available this will be addressed. during this study, we identified three additional species that possibly have two tor paralogs: m. brevicollis, thecamonas trahens, and allomyces macrogynus, species that are included in the origins of multicellularity project. while analysis is limited because of the state of the m. brevicollis genome, it appears that an additional bonafide tor protein is encoded with the conserved tor protein architecture. thecamonas trahens is a unicellular apusomonad that also contains two tor paralogs. interestingly, a. macrogynus is a fungus that may contain a second paralog; however, it appears that the frb domain overlaps with the kinase domain. this may represent a case in which tor was duplicated but is in the process of losing one of the copies. these cases are a springboard for future studies involving the duplication of tor within and beyond the fungal kingdom. loss of the tor signaling pathway in microsporidia while components of the tor signaling cascade are conserved in eukaryotes from yeasts to humans, the microsporidia, a group of highly specialized obligate intracellular pathogens completely lack all of the pathway components investigated in this study. e. cuniculi has previously been noted to lack the tor kinase, and we found that all pathway components including tor were missing not only in e. cuniculi but also in its microsporidian relatives e. bieneusi and n. ceranae. microsporidian genomes are highly compacted and show a marked reduced gene content. the genome of the human pathogen e. cuniculi has genes distributed throughout chromosomes in a mb genome, less than half the number of protein-coding genes found in escherichia coli. for comparison, the genome of the human pathogen e. bieneusi genome is ~ mb with genes and the honeybee pathogen n. ceranae genome is ~ mb with predicted genes. genome reduction has been well studied in intracellular bacteria: during the specialization process from a free-living to intracellular lifestyle, massive gene loss can occur. we hypothesize that as a consequence of their specialization to an obligate intracellular lifestyle, the need for nutrient sensing was relaxed and these obligate intracellular microsporidia acquired nutrients from within the host cytoplasm. thus, they no longer required the tor pathway, which is dedicated to sensing changes in the nutrient composite of the environmental milieu. however, e. cuniculi has retained some of the genes encoding signaling pathways involved in nutrient sensing in other species, including homologs of s. cerevisiae ras and ras. interestingly, p. carinii is an obligate pathogen with a reduced genome estimated to be ~ mb, which unlike microsporidia is not obligately intracellular. a single tor homolog was identified using the s. cerevisiae tor frb domain in tblastx, supporting our hypothesis that the microsporidia have streamlined their genomes as they adopted an obligate intracellular life cycle. p. carinii likely requires tor because it must survive in a metabolically active form outside of host cells, undergoing cell division in the extracellular milieu of the infected host lung. many microsporidian genomes have undergone extreme genome reduction and are among the eukaryotes with the smallest genomes. in addition to fewer genes than other species, e. cuniculi has shorter genes, small intergenic distances, and very few introns. this species lacks several genes that are necessary for biosynthetic pathways and the tricarboxylic acid cycle in free-living organisms. remarkably, e. cuniculi must acquire atp from its host through a series of atp transporters anciently acquired from rickettsia- or chlamydia-like bacteria, and this is an example of how e. cuniculi, and possibly other microsporidia, can hijack mechanisms of other intracellular bacteria, fungi, or the host to survive in and adapt to their highly specialized intracellular lifestyle.  the tor pathway is highly conserved with some exceptions among fungi, including several pathogens. similarly, the molecular structural organization of the tor kinases has been remarkably conserved as well as the presumed ability to bind the fkbp12-rapamycin complex. this feature, combined with the essential nature of the rapamycin-sensitive torc pathway for cell growth, should enable the development of rapamycin-based strategies for antifungal therapies. duplications of the tor protein occurred in most fungal groups examined, resulting from either independent segmental gene duplication events or a wgd event. the maintenance of two tor homologs can be explained through the ddc model of gene duplication, in which the paralogs subfunctionalize and together support all of the functions of the pre-duplicated protein. in addition, species outside of the fungal kingdom with two or more tor homologs include the metazoan b. mori, the apusomonad protozoan t. trahens, the trypanosome protozoans l. major and t. brucei, and possibly the choanoflagellate m. brevicollis. this suggests that the tor gene has been independently duplicated multiple times since the last common ancestor of the opisthokonta and excavata lineages. the tor pathway, as well as the tor protein itself, is highly conserved in eukaryotes, so the observation that three microsporidian species are missing the entire pathway is striking. it would appear that the specialized obligate intracellular life cycle of microsporidia allows for the acquisition of nutrients from the host cell, obviating a requirement for this central nutrient-sensing pathway that is essential in all known cases of other eukaryotes for axenic growth. 
capitalizing on the plethora of recently sequenced genomes we have conducted comparative genomic studies to annotate the tor pathway throughout the fungal kingdom and related unicellular opisthokonts, including monosiga brevicollis, salpingoeca rosetta, and capsaspora owczarzaki. the nutrient-sensing tor pathway governs cell growth and is conserved in nearly all eukaryotic organisms from unicellular yeasts to multicellular organisms, including humans. the microsporidia are obligate intracellular pathogens with highly reduced genomes, and we hypothesize that they lost the tor pathway as they adapted and streamlined their genomes for intracellular growth in a nutrient-rich environment. interestingly, the tor signaling cascade is absent in three microsporidian species with available genome sequences, the only known instance of a eukaryotic group lacking this conserved pathway. the repeated independent duplications of the tor gene in the fungal kingdom might reflect selective pressure acting upon this kinase that populates two proteinaceous complexes with different cellular roles. two tor paralogs are present in several fungal species as a result of either a whole genome duplication or independent gene/segmental duplication events. these comparative genomic analyses illustrate the evolutionary trajectory of a central nutrient-sensing cascade that enables diverse eukaryotic organisms to respond to their natural environments.
 the nutrient-sensing tor pathway governs cell growth and is conserved in nearly all eukaryotic organisms from unicellular yeasts to multicellular organisms, including humans. tor is the target of the immunosuppressive drug rapamycin, which in complex with the prolyl isomerase fkbp inhibits tor functions. rapamycin is a gold standard drug for organ transplant recipients that was approved by the fda in and is finding additional clinical indications as a chemotherapeutic and antiproliferative agent. capitalizing on the plethora of recently sequenced genomes we have conducted comparative genomic studies to annotate the tor pathway throughout the fungal kingdom and related unicellular opisthokonts, including monosiga brevicollis, salpingoeca rosetta, and capsaspora owczarzaki.  interestingly, the tor signaling cascade is absent in three microsporidian species with available genome sequences, the only known instance of a eukaryotic group lacking this conserved pathway. the microsporidia are obligate intracellular pathogens with highly reduced genomes, and we hypothesize that they lost the tor pathway as they adapted and streamlined their genomes for intracellular growth in a nutrient-rich environment. two tor paralogs are present in several fungal species as a result of either a whole genome duplication or independent gene/segmental duplication events. one such event was identified in the amphibian pathogen batrachochytrium dendrobatidis, a chytrid responsible for worldwide global amphibian declines and extinctions.  the repeated independent duplications of the tor gene in the fungal kingdom might reflect selective pressure acting upon this kinase that populates two proteinaceous complexes with different cellular roles. these comparative genomic analyses illustrate the evolutionary trajectory of a central nutrient-sensing cascade that enables diverse eukaryotic organisms to respond to their natural environments. 