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Frequent, independent transfers of a
catabolic gene from bacteria to contrasted
filamentous eukaryotes
rspb.royalsocietypublishing.org
Maxime Bruto1,2,3, Claire Prigent-Combaret1,2,3, Patricia Luis1,2,3,
Yvan Moënne-Loccoz1,2,3 and Daniel Muller1,2,3
1
Université de Lyon, Lyon 69622, France
Université Lyon 1, Villeurbanne, France
3
CNRS, UMR5557, Ecologie Microbienne, Villeurbanne, France
2
Research
Cite this article: Bruto M, Prigent-Combaret
C, Luis P, Moënne-Loccoz Y, Muller D. 2014
Frequent, independent transfers of a catabolic
gene from bacteria to contrasted filamentous
eukaryotes. Proc. R. Soc. B 281: 20140848.
http://dx.doi.org/10.1098/rspb.2014.0848
Received: 9 April 2014
Accepted: 5 June 2014
Subject Areas:
evolution, ecology
Keywords:
inter-domain transfer, lateral gene transfer,
adaptive horizontal transfer
Author for correspondence:
Daniel Muller
e-mail: [email protected]
Electronic supplementary material is available
at http://dx.doi.org/10.1098/rspb.2014.0848 or
via http://rspb.royalsocietypublishing.org.
Even genetically distant prokaryotes can exchange genes between them, and
these horizontal gene transfer events play a central role in adaptation and evolution. While this was long thought to be restricted to prokaryotes, certain
eukaryotes have acquired genes of bacterial origin. However, gene acquisitions
in eukaryotes are thought to be much less important in magnitude than in
prokaryotes. Here, we describe the complex evolutionary history of a bacterial
catabolic gene that has been transferred repeatedly from different bacterial
phyla to stramenopiles and fungi. Indeed, phylogenomic analysis pointed to
multiple acquisitions of the gene in these filamentous eukaryotes—as many
as 15 different events for 65 microeukaryotes. Furthermore, once transferred,
this gene acquired introns and was found expressed in mRNA databases for
most recipients. Our results show that effective inter-domain transfers and
subsequent adaptation of a prokaryotic gene in eukaryotic cells can happen
at an unprecedented magnitude.
1. Introduction
In nature, species need to constantly adapt to changing environments, and this
can be achieved by modifying their genetic repertoire to acquire new functions.
Indeed, gene duplications (followed by evolution of new functions) and other
genomic rearrangements have shaped eukaryotic genomes [1]. However, genetic innovation can also result from the acquisition of exogenous genes by
horizontal gene transfer (HGT). Prokaryotes adapt largely by HGT, and strains
of a particular species can differ by large fractions of their genome [2–6].
Long thought to be a prokaryote specialty, HGT is now recognized as a mechanism of genetic innovation in eukaryotes as well [7–10]. Genome analysis of
eukaryotes revealed that several genes had been horizontally transferred [11,12],
with important implications for environmental adaptation [13–15]. Indubitably,
HGT can enable acquisition of entirely novel functions, which is more drastic
than the gradual evolutionary processes that rely on modification of pre-existing
genes [16–18] and may enhance ecological opportunities. In this context, the prokaryotic gene pool can serve as a large reservoir of potential functions for
eukaryotes [9]. Indeed, it appears that prokaryote-to-eukaryote inter-domain
HGT events are more prevalent than eukaryote-to-eukaryote ones [19]. As
described for the insect Hypothenemus hampei, where inter-domain HGT of a mannase-encoding gene from a Firmicute enabled the insect to parasitize coffee berries
[7], the acquisition of a single gene can lead to enhanced competitiveness and ecological specialization. However, inter-domain HGT can involve more than one
gene. Thus, previous studies reported that significant parts of genome, up to 10%
of the gene repertoire of the multicellular rotifer Adineta ricciae [13], had been horizontally acquired. Although these acquisitions resulted from several transfers,
the extent to which a given bacterial gene may undergo inter-domain transfers to
eukaryotes remains unclear.
& 2014 The Author(s) Published by the Royal Society. All rights reserved.
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(a) acdS prevalence in eukaryotes
We found as many as 65 acdS homologues in eukaryotes—four
in stramenopiles (all oomycetes) and 61 in fungi—after analysis
of 149 sequenced genomes based on the AcdS protein sequence
of the proteobacterium Pseudomonas fluorescens F113. Significant
sequence identity (at least 38% amino acid identity) was evidenced between bacterial and eukaryotic AcdS proteins.
Moreover, conservation of nucleotide sequences between bacterial and eukaryotic acdS genes was also high (at least 40%
identity). It reached as much as 65% between the actinobacterium Streptomyces violaceusniger Tu4113 and the ascomycotan
M. oryzae 70–15, and 78% between the proteobacterium
Acidovorax radicis N35 and the stramenopile Phytophthora
infestans T30-4. Such gene sequence conservation across two
different domains of life suggests both a common evolutionary
origin and genetic transfer(s) between them.
(b) Distribution of acdS homologues in eukaryotes
Phylogenetic analysis of AcdS protein homologues retrieved by
BLAST showed that eukaryotic AcdS sequences were distributed in three distinct clades (figure 1; electronic supplementary
material, figures S1–S3). The first clade is rooted by actinobacterial AcdS sequences and corresponds exclusively to fungi
(Ascomycota and a few Basidiomycota). The second clade is
rooted by gammaproteobacterial AcdS sequences and only
includes Ascomycota. The third clade is rooted by betaproteobacterial AcdS sequences, and includes a stramenopile
subclade (Phytophthora species) and a fungal subclade. This topology was retrieved both with maximum-likelihood and
Bayesian reconstructions [26] (see the electronic supplementary
(c) Estimated acdS gains and losses along fungal
evolutionary history
To assess the extent of acdS transfer and loss events, we reconstructed the ancestral states of acdS presence/absence along
fungal phylogenetic history, using 150 sequenced fungi. For actinobacterial donors, ancestral state reconstruction showed four
supported acquisitions in Basidiomycota, which concerned
Gymnopus luxurians, Schizophyllum commune, Gloeophyllum
trabeum (Agaricomycetes class) and a Cryptococcus ancestor (Tremellomycetes class) (figure 2). Similarly, four acquisitions were
identified for Ascomycota: a recent one in Oidiodendron maius
(Leotiomycetes class), as well as three more ancient ones in the
Eurotiomycetes class ancestor, in the Sordariomycetes class
ancestor and in a Mycosphaerellaceae subclade (Dothideomycetes class). Ancestral state reconstruction for the acdS clade of
betaproteobacterial origin pointed to two recent acdS acquisitions in the Basidiomycota Fomitopsis pinicola SS1 and
Punctularia strigosozonata (Agaricomycetes class), and another
acquisition by an unidentified ancestor of the Dothideomycetes
Hysterium pulicare, Rhytidisteron rufulum and Botryosphaeria dothidea. Complete reconstructions for these two clades can be found
in the electronic supplementary material, figures S4 and S5.
For gammaproteobacterial acdS donors, ancestral state
reconstruction did not provide a clear scenario using
maximum-likelihood reconstruction (see the electronic supplementary material, figure S6), but Bayesian reconstruction
strongly pointed to independent acquisitions in every acdSþ
fungus (see the electronic supplementary material, table S1).
Yet these two reconstructions strongly support the absence of
acdS in the most ancestral nodes for the Saccharomycetes, refuting the hypothesis of a single, ancestral acquisition of the gene.
For each putative ancestral recipient, no case of subsequent
acdS gene loss was detected in the descent, regardless of
whether the gene originated from Actinobacteria, Betaproteobacteria or Gammaproteobacteria. Thus, our results point to
recurrent HGT events of acdS towards oomycetes and fungi.
(d) Functionality and selection patterns of
eukaryotic acdS
Functionality of eukaryotic acdS is indicated by the conservation of the catalytic function. Indeed, the key residues K51,
Y269, Y295 and E296 needed for ACC deaminase catalytic
activity in the yeast C. saturnus [23] were conserved in all
eukaryote sequences (both in Phytophthora and fungi), as
were the amino acids adjacent to these residues (figure 3).
This highlights the conservation of the bacterial acdS catalytic
function across distant eukaryotic lineages that experienced
independent acdS acquisitions. In addition, direct evidence
for acdS transcription in eukaryotes was also obtained, as
2
Proc. R. Soc. B 281: 20140848
2. Results
material, figure S1). Thus, our results strongly support a bacterial origin for acdS eukaryotic homologues, and also indicate
that Actinobacteria, Betaproteobacteria and Gammaproteobacteria served as distinct acdS donors for eukaryotes. Fungal
recipients belonged to several taxonomical classes, and different
possibilities may account for the uneven distribution of acdS
homologues in oomycetes and fungi. The first hypothesis is
an ancestral acquisition of the gene followed by multiple
losses in a broad range of eukaryotic lineages. The second
hypothesis entails multiple HGT events, perhaps even between
different types of eukaryotes.
rspb.royalsocietypublishing.org
The bacterial gene acdS has been evidenced not only in taxonomically contrasted bacteria [20], notably in strains with plant
growth-promotion activity, but also in a few fungi [21–23].
The AcdS enzyme 1-aminocyclopropane-1-carboxylate (ACC)
deaminase (EC 4.1.99.4) can transform the plant’s ethylene
precursor ACC to a-ketobutyrate and ammonia. By degrading
ACC in exudates, plant-interacting bacteria can indirectly
lower ethylene level in roots, thus stimulating root growth and
modulating plant stress resistance [24,25]. In addition to the
acdS gene itself, AcdS catalytic activity was also found in fungi,
namely the Ascomycota Trichoderma asperellum [22], Cyberlindnera (formerly Hansenula) saturnus [23], Penicillium citrinum [21]
and Magnaporthe oryzae (M.B., C.P.-C., P.L., Y.M.-L. & D.M.
2013, unpublished data), raising the question of the evolutionary
origin of this gene in eukaryotes.
In this study, we show that the bacterial gene acdS has
been repeatedly transferred to a wide range of eukaryotic
recipients (i.e. fungi and stramenopiles). Phylogenetic analysis pointed to multiple acdS acquisitions, from different
bacterial phyla to different eukaryotes. Ancestral state character reconstruction confirmed past occurrence of multiple,
independent transfers of acdS from each of these bacterial
phyla to different types of eukaryotes. Moreover, transferred
acdS genes were effectively transcribed and occasionally
acquired introns in eukaryotic recipients. Altogether, these
results show that prokaryote-to-eukaryote transfer of a
single gene can happen at high frequency, with adaptation
of the transferred gene to its new host cell machinery.
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0.1
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Pseudomonas psychrotolerans L19
Herbaspirillum seropedicae SmR1
Meiothermus ruber DSM 1279
Alphaproteobacteria
Achromobacter
Cupriavidus basilensis OR16
Burkholderia cenocepacia PC184
Cupriavidus necator N 1
Ralstonia eutropha H16
Burkholderia
Betaproteobacteria
Collimonas fungivorans Ter331
Variovorax paradoxus S110
Variovorax paradoxus EPS
Acidovorax
Ralstonia
Proc. R. Soc. B 281: 20140848
Hysterium pulicare
Rhytidhysteron rufulum
Punctularia strigosozonata
Fomitopsis pinicola SS1
Botryosphaeria dothidea
Phytophthora ramorum
Phytophthora sojae
Phytophthora capsici
Phytophthora infestans T30 4
Acidovorax
Polaromonas sp JS666
Methylibium petroleiphilum PM1
Betaproteobacteria
Ralstonia
Halomonas
Pseudomonas
Dickeya dadantii Ech703
Dickeya
Gammaproteobacteria
Brenneria sp EniD312
Pantoea sp At 9b
Pachysolen tannophilus NRRLY2460
Schizosaccharomyces pombe 972h
Bajeviella inositovora NRRLY12698
Clavispora lusitaniae ATCC42720
Pichia membranifaciens
Actinobacteria
Chromalveolata (kingdom) :
- Oomycetes
fungi (kingdom) :
+ Basidiomycota (phylum) :
- diverse taxonomic ranks
+ Ascomycota (phylum) :
- Dothideomycetes (class)
- Eurotiomycetes (class)
- Saccharomycetes/Schizosaccharomyces (class)
- Sordariomycetes (class) :
• Mycosphaerellaceae (family)
• Magnaporthaceae (family)
• Chaetomiaceae (family)
• Plectosphaerellaceae (family)
• Cryphonectriaceae (family)
• Hypocreales (family)
• Sordariaceae (family)
• Lasiosphaeriaceae (family)
• Myxotrichaceae (family)
Mycosphaerella graminicola
Gleophyllum trabeum
Cryptococcus neoformans B 3501A
Cryptococcus neoformans JEC21
Schizophyllum commune
Mycosphaerella fijiensis
Dothistroma septosporum NZE10
Cercospora zeae maydis
Septoria musiva S02202
Septoria populicola
Coccidioides immitis RS
Coccidioides posadasi C735 delta SOWgp
Trichophyton rubrum CBS118892
Arthroderma benhamiae CBS112371
Trichophyton verrucosum HK10517
Arthroderma gypseum CBS118893
Arthroderma otae CBS113480
Penicillium chrysogenum
Aspergillus aculeatus ATCC16872
Aspergillus flavus NRRL3357
Aspergillus oryzae RIB40
Neosartorya fischeri NRRL181
Aspergillus carbonarius ITEM 5010
Aspergillus niger
Gymnopus luxurians
Chaemotium globosum
Thielavia terrestris
Sporotrichum thermophile
Podospora anserina Smat
Sordaria macrospora k hell
Neurospora crassa OR74A
Neurospora discreta FGSC8579
Neurospora tetrasperma FGSC2508
Neurospora tetrasperma FGSC2509
Acremonium alcalophilum
Magnaporthe grisea
Penicillium marneffei ATCC18224
Aspergillus nidulans
Oidiodendron maius
Verticillium dahliae
Cryphonectria parasitica EP155
Nectria hematococca
Fusarium oxysporum
Fusarium graminearum
Gibberella zeae PH 1
Trichoderma reesei
Trichoderma reesei RUTC 30
Trichoderma harzianum CBS226 95
Trichodemna virens Gv29 8
Trichoderma asperellum CBS433 97
Trichoderma atroviridae
Figure 1. Maximum-likelihood phylogenetic tree of acdS protein sequences. The tree was rooted using D-cysteine desulfhydrase sequences as outgroup (electronic
supplementary material, figure S3). Supported nodes are indicated with grey circles (bootstrap . 70).
mining transcript databases allowed the identification of all
or part of acdS mRNA in almost all stramenopiles and
fungi studied (see the electronic supplementary material,
figure S7). This indicated that differences in promoter regions
between bacteria and eukaryotes were not a barrier for
successful genetic transfers.
The lack of acdS deletion and the high AcdS sequence
conservation suggest that this gene confers a selective advantage to microeukaryotes. In addition, comparing relative
fixation rates of synonymous (silent) and non-synonymous
(amino acid altering) mutations showed strong negative
selection (dN/dS ratio , 1) in most species (see the electronic
supplementary material, figure S8), meaning that functional
mutational modifications were selected against. Despite this
purifying selection, positive selection (dN/dS ratio . 1) was
also found in ancestral branches, notably in the Ascomycota
Aspergillus (Eurotiomycetes class), Trichoderma and Fusarium
(Sordariomycetes class), and in the Dothideomycetes class.
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Figure 2. Ancestral state character reconstruction of acdS gene gains/losses. Coloured nodes and species names indicate gene presence while black means absence.
Actinobacterial, betaproteobacterial and gammaproteobacterial acdS origins are shown in, respectively, red, blue and green for these names and nodes. Uncertainty
in reconstruction is indicated by a white node. Fungal classes: (1) Chytridiomycetes; (2) Monoblepharidomycetes; (3) Neocallimastigomycetes; (4) Entomophthoromycotina; (5) Kickxellomycotina; (6) Mucoromycotina; (7) Exobasidiomycetes; (8) Ustilaginomycetes; (9) Microbotryomycetes; (10) Mixiomycetes;
(11) Pucciniomycetes; (12) Wallemiomycetes; (13) Tremellomycetes; (14) Dacrymycetes; (15) Agaricomycetes; (16) Taphrinomycotina; (17) Schizosaccharomycetes;
(18) Saccharomycetes; (19) Pezizomycetes; (20) Leotiomycetes; (21) Sordariomycetes; (22) Lecanoromycetes; (23) Eurotiomycetes; (24) Teratosphaeriaceae;
(25) Mycosphaerellaceae; (26) Dothideomycetes.
This diversifying selection, which means that functional AcdS
modifications could be selected, is more likely to reflect
sequence adaptation to gene biology in eukaryotes rather
than a true change in protein function. This hypothesis is
strengthened by the conservation of key residues implicated
in the catalytic function of the protein, as well as demonstration
of AcdS enzymatic activity in fungi tested [21–23].
(e) Intron acquisitions in eukaryotic acdS sequences
Unlike in prokaryotes, eukaryotic genes typically display a
combination of introns and exons, and indeed we found
one or several spliceosomal introns (up to eight in the
Basidiomycota G. trabeum) in eukaryotic acdS sequences
(figure 4). Thus, acdS acquisition by eukaryotes was followed by intron formation(s) in around half the identified
transfers (31 of 65 eukaryotes). Most Sordariomycetes (corresponding to nine distinct taxonomic families) presented a
conserved region of intron insertion, located 186–195 nucleotides from the acdS start codon. Introns were also found in
this region in species belonging to distant taxonomic classes,
such as the Lecanoromycete O. maius Zn and the Eurotiomycete Penicillium marneffei ATCC18224. However, the intron
sequences themselves showed no conservation, except in closely related species (data not shown). Taken together, the
data point to lineage-specific intronization, which might
have gone on par with acdS evolution and domestication
within these lineages.
Proc. R. Soc. B 281: 20140848
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Figure 3. Conservation of key amino acids in acdS. Conserved amino acids are shown in (a) the Lys region, (b) the Tyr region and (c) the Tyr294-Glu295 region,
which are important for catalytic activity in bacterial and eukaryotic AcdS sequences [23]. Positions indicated refer to those in the protein sequence of the
Ascomycota model C. saturnus, for which key residues (boxed) were identified.
HGT is a key feature of bacterial evolution [27,28], but recent
studies have also reported HGT events from prokaryotes to
eukaryotes [3,7–10,12]. A significant portion of the genome of
certain eukaryotic species was acquired horizontally [13–15],
yet it was not clear at which order of magnitude such transfers
could take place for a given gene. This work demonstrates that
the bacterial gene acdS is extensively present in filamentous
eukaryotes, based on the recovery of this gene in as many as
44% of the 149 sequenced genomes available. Different processes can explain the uneven distribution of genes in a given
lineage, such as convergent evolution [29,30], lineage-specific
gene loss [31,32] or HGT [8,33,34]. In our case, convergent evolution is unlikely as acdS nucleotide sequence identity of
bacterial and eukaryotic homologues (40–78%) is particularly
high, indicating a similar evolutionary origin.
Even though the gene loss hypothesis cannot be fully
rejected, data clearly point to multiple and independent HGT
events for acdS. In particular, eukaryotic acdS homologues do
not form an independent clade outside bacterial acdS homologues (as expected if genes were vertically transmitted) but
are rather interspersed within bacterial sequences. Thus, acdS
phylogeny identified three major incongruences with the
species tree (electronic supplementary material, figure S2),
indicating HGT from three bacterial phyla to many eukaryotic
lineages. Moreover, incongruences were also found inside
eukaryotic clades, suggesting eukaryote-to-eukaryote transfers. Such events can take place [31,34], but here poor node
supports failed to strengthen the hypothesis.
For two of the three major incongruences—namely those
involving (i) branching of various fungi and stramenopiles
(two distant eukaryotic lineages [35,36]) inside Betaproteobacteria and (ii) Basidiomycota and Ascomycota in relation to
Actinobacteria—this could entail a unique acquisition of acdS
by a eukaryotic ancestor followed by extensive gene loss
during evolution and speciation of the different eukaryotic
lineages. Considering vertical transfer to explain the patterns
of acdS presence would imply an ancestral acquisition in an
ancestor common to various eukaryotes. However, this
hypothesis is rather unlikely considering the high conservation of acdS sequences in the eukaryotic lineages in each
case. An alternative possibility is a series of multiple, distinct
acdS transfers to different microeukaryotic clades. The two
ancestral character reconstruction methods that were used
clearly pointed to the latter possibility, which is very well supported statistically when considering acdS distribution in
relation to fungal and stramenopile evolutionary histories.
The third incongruence involves Gammaproteobacteria
and diverse Saccharomycetes/Schizosaccharomycetes. In this
case, the two methods of ancestral character reconstructions
gave conflicting results, thus inferences should be taken
with caution. Maximum-likelihood reconstruction pointed to
a unique, ancestral acquisition of acdS followed by subsequent
vertical transmission and differential gene losses. However,
10 of 16 nodes of the Saccharomycetes/Schizosaccharomycetes
clade were not statistically supported, limiting conclusions
on the current analysis. By contrast, Bayesian reconstruction inferred the presence of the gene in a last eukaryotic
common ancestor, but this was unlikely (and thus might
reflect analysis bias) because only a few Saccharomycetes/
Schizosaccharomycetes fungi possessed acdS. Nevertheless, a
robust Bayesian reconstruction was obtained when constraining the model to infer the absence of acdS at the root of the
tree. In this case, the analysis favours multiple and independent transfers with strong statistical support at each node of
the Saccharomycetes/Schizosaccharomycetes clade.
In the 65 acdS þ microeukaryotes, we estimated that acdS
acquisition entailed at least 15 different HGT events based
on ancestral character reconstructions (figure 2). Such a magnitude for genetic transfer of a bacterial gene across a vast
range of filamentous eukaryotes has never been described
before [37,38] and challenges our understanding of eukaryote
evolution, as most current models in evolutionary biology
assume gene duplication as a key process of biochemical
innovation [39]. In contrast to duplication, which gives rise
to slow genetic innovation, gene acquisition might play a
distinct role in enabling rapid phenotypic or ecological adaptation. Nevertheless, the actual mechanism(s) by which acdS
was acquired by filamentous eukaryotes remain(s) unknown.
We did not find any remnant of a mobile genetic element in the
vicinity of acdS insertion sites. Furthermore, other putative
bacterial genes in the vicinity of acdS were not found in eukaryotic genomes based on sequence identity search, except one
encoding a putative monooxygenase downstream acdS in
Eurotiomycetes, but that is also largely present in acdS-negative
fungi (data not shown). Thus, it seems that acdS could have
been transferred alone. Phagotrophy, the consumption of a
whole cell by another one, is seen as a driving force in
bacteria-to-unicellular eukaryote HGT [40], but cannot explain
HGT in fungi [41].
The fact that most acdS þ microeukaryotes live in the vicinity
of plants is of primary interest, because sharing a same ecological habitat may facilitate physical interaction between HGT
protagonists [42,43] and is likely to promote acdS transfer. In
addition, plants are the main natural source of ACC, which
may represent a significant source of carbon (a-ketoglutarate)
and nitrogen (ammonia) for AcdSþ plant-associated eukaryotes
[44]. In return, the latter act on plants by decreasing ethylene
levels. In non-pathogenic fungi like T. asperellum T203, this
Proc. R. Soc. B 281: 20140848
3. Discussion
268
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probability
(a)
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(a)
(b)
0.01
(c)
Punctularia strigosozonata
Fomitopsis pinicola SS1
Botryosphaeria dothidea
Hysterium pulicare
Rhytidhysteron rufulum
Phytophthora infestans T30-4
Phytophthora capsici
Phytophthora sojae
0.01
Phytophthora ramorum
(d)
Schizosaccharomyces pombe
Pachysolen tannophilus NRRL Y–2460
Pichia membranifaciens
Babjeviella inositovora NRRL Y–12698
0.01
Clavispora lusitaniae
Figure 4. Genetic structure of eukaryotic acdS in relation to the position in the acdS phylogenetic gene tree. Coding sequences are represented by white rectangles
and introns by black lines. (a) Fungal acdS of actinobacterial origin, (b) fungal acdS of betaproteobacterial origin, (c) Phytophthora acdS of betaproteobacterial origin
and (d ) fungal acdS of gammaproteobacterial origin.
promotes root elongation of canola and cucumber, favouring
plants’ nutrient uptake and in turn their growth and yields
[22,45]. In phytopathogenic oomycetes and fungi, however,
the main effect of ACC degradation might be to facilitate
plant infection, because ethylene signalling acts synergistically
with jasmonate to induce plant defence responses [46,47].
The ACC deamination case is unusual in that the function
can be ecologically important for microeukaryotes (and thus
Proc. R. Soc. B 281: 20140848
0.01
6
rspb.royalsocietypublishing.org
Cryptococcus neoformans var JEC21
Cryptococcus neoformans
Gloeophyllum trabeum
Schizophyllum commune
Gymnopus luxurians
Mycosphaerella graminicola
Dothistroma septosporum NZE10
Mycosphaerella fijiensis
Cercospora zeae-maydis
Septoria musiva SO2202
Septoria populicola
Coccidioides immitis
Coccidioides posadasii
Arthroderma otae
Arthroderma gypseum
Trichophyton rubrum
Arthroderma benhamiae
Trichophyton verrucosum
Penicillium marneffei
Neosartorya fischeri
Aspergillus nidulans
Penicillium chrysogenum
Aspergillus flavus
Aspergillus oryzae
Aspergillus aculeatus ATCC16872
Aspergillus carbonarius ITEM 5010
Aspergillus niger
Oidiodendron maius Zn
Cryphonectria parasitica EP155
Magnaporthe oryzae
Podospora anserina
Chaetomium globosum
Thielavia terrestris
Sporotrichum thermophile
Sordaria macrospora
Neurospora discreta FGSC 8579
Neurospora crassa OR74A
Neurospora tetrasperma FGSC 2509
Neurospora tetrasperma FGSC 2508
Acremonium alcalophilum
Verticillium dahliae
Nectria haematococca
Fusarium oxysporum
Fusarium graminearum
Gibberella zeae
Trichoderma asperellum CBS 43397
Trichoderma atroviride
Trichoderma reesei
Trichoderma reesei RUT C–30
Trichoderma harzianum CBS 226 95
Trichoderma virens Gv29–8
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(a) Homologous sequence retrieval and re-annotation
The AcdS protein sequence (YP_005208895) of P. fluorescens F113
was queried with BLASTp [48] against the NCBI RefSeq database
[49] to retrieve prokaryotic homologues, with an E-value threshold
of 110220 to filter results. To retrieve eukaryotic homologues,
the same sequence was simultaneously queried with BLASTp
against the JGI Mycocosm [50] and NCBI RefSeq databases, with
same 110220 E-value threshold. ACC deaminase sequences
were further selected among the retrieved homologues based on
functional domain identification, using RPS-BLAST [48].
(b) Phylogenetic analysis
Protein sequences were aligned with CLUSTAL OMEGA [51].
Sequences were manually filtered to discard gaps and aligned
regions of low quality. For acdS sequences, the phylogenetic
trees were inferred with PHYML [52] with the GTR model, 1000
bootstraps, SPR topology search [53] and the estimation of the
proportion of invariant sites. Paralogues were identified by a
phylogenetic approach and removed from the analysis.
(c) Fungal phylogenetic tree reconstruction
The fungal species tree was inferred from 43 concatenated protein
markers [54]. For the latter, the markers were obtained for 145
sequenced fungi using HMMER3. When multiple homologues of
a given marker were retrieved for a given fungal species, redundancy was resolved using a tree-based approach, and markers
(d) Ancestral state reconstruction
Maximum-likelihood and Bayesian reconstructions were used to
mitigate potential methodological biases of each approach
[58,59]. The maximum-likelihood method allows quick reconstruction of ancestral state with a good sensitivity [59], whereas Bayesian
methods of reconstruction have the advantage of taking into
account tree uncertainty by reconstructing ancestral states over a
set of phylogenetic trees [60]. Analyses were done with the previously inferred fungal tree and matrices of presence/absence of
fungal acdS homologues, using MESQUITE (http://mesquitepro
ject.org/mesquite/mesquite.html) with maximum likelihood
Asymmk2 model of rate variations [61] and the reversible jump
Markov chain Monte Carlo method of BAYESTRAITS [60]. Statistical
confidence was assessed with a likelihood ratio test and the
Bayes factor ratio, respectively.
(e) Conservation of key amino acids in eukaryotic
acdS homologues
Unfiltered alignments were used to verify the presence of amino
acids required for ACC deaminase activity. Structural analysis
along with site-directed mutational studies of the AcdS protein
in C. saturnus identified four important amino acids (K51, Y269,
Y295 and E296 [23]). Multiple alignments were numbered according to C. saturnus sequence and represented using WEBLOGO
(http://weblogo.berkeley.edu).
(f ) Intron annotation
Introns were re-annotated in nucleotide sequences using WISE
v. 2.1.20 (http://www.ebi.ac.uk/Tools/psa/genewise). A hidden
Markov model (HMM) profile was generated using HMMER3
and served as query to align against nucleotide sequences. WISE
software was set to consider GT/AG splicing sites only.
Acknowledgements. We thank V. Daubin, N. Carraro and S. Chuzeville for
useful discussions, D. Abrouk and A. Dubost (iBio) for technical support,
G. Hoff and M-A. Poirier for fungal assays and the P2CHPD (Pôle de
Calcul Haute Performance Dédiées) for access to computing facilities.
Funding statement. This work was supported by the Ministère Français
de la Recherche.
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