Download NRT2 - Clark University

Molecular evolution of nrt2, a nitrate transporter gene, with an emphasis on
Hebeloma phylogeny
Jason C. Slot and David S. Hibbett
Department of Biology, Clark
University, Worcester, MA 01610
Presented at the Joint Meeting of the
Mycological Societies of America and
Japan, August, 2005 in Hilo, HI, USA
[email protected], [email protected]
Clockwise from upper left Gymnopilus sp., Hebeloma velutipes, Alnicola escharoides, Hebeloma sp.
Courtesy of P.B. Matheny
Two copies of nrt2: Above is a schematic representation of the NRT2 protein
Introduction:
The most ephemeral source of nitrogen in the soil, nitrate, is
transported into cells by nitrate transporters of differential affinities for
nitrate. A nitrate assimilation gene cluster containing a high affinity nitrate
transporter gene, nrt2 along with nitrate and nitrite reductase genes was
recently characterized in Hebeloma cylindrosporum.
NO3- (environmental) --> NO3- (cellular) --> NO2- --> NH4+ -->Organic
Nitrogen
NRT2
NAR1 NIR1
Hebeloma cylindrosporum is an ectomycorrhial basidiomycete
belonging to a clade that is characterized by a diversity of soil nitrogenstatus preferences. For example, H. radicosum forms a tripartite symbiosis
involving mole latrines and H. syrjense is appropriately called “corpefinder”, while H. cylindrosporum is found in sand and many other
Hebeloma prefer nitrogen-poor acidic soil. In addition to being
chemoecologically diverse, Hebeloma has been problematic for traditional
and molecular systematists alike. Hebeloma, furthermore appears to be an
example of a recent acquisition of mycorrhization, or a symbiotic
stronghold within a clade of many mycorrhiza-losers (such as Agrocybe,
Hypholoma and Gymnopilus).
This study explores the evolution of nrt2 sequences relative to that
of the Internal Transcribed Spacer, ITS, sequences of nuclear rDNA.
Patterns in nitrate-assimilation gene evolution could improve the
understanding of the selective pressure exerted by soil nitrogen availability,
and the relative significance of nitrate in the evolution of Ectomycorrhizal
relationships.
NR -
sampled for nrt
H .edurum C BS291.50 NR
H . sinuosum C BS184.47
NR
H ebeloma edurum 637
H ebeloma dani cum LY64BR 38
60
H ebeloma cyl indr osporum 6100
H .cyl indr osporum CBS558.96 NR
H ebeloma bi rrus 580
H ebeloma r adicosum 640
nitrophili c
H .radicosum CB S183.47 NR
H ebeloma ci ricnans 638
H ebeloma si napizans 514
H ebeloma truncatum 641
H ebeloma sp.PBM2693c3
H ebeloma sp.PBM2693c2 NR
H ebeloma sp. JC S91904A NR
H ebeloma sp. PBM2691
59
H ebeloma sacchari olens 552
H ebeloma tomentosum 506
H . truncatum CBS295.50
NR
2
H ebeloma vel uti pes 5041
H ebeloma vel uti pes 535
H ebeloma vel uti pes 642
H ebeloma i ncarnatul um 527
H ebeloma vel uti pes 540
H . velutipes UP181
H. velutipesclad
e
H ebeloma vel uti pes AFTOL
H ebeloma vel uti pes 502
H ebeloma vel uti pes 5042
86
H .vel uti pes C BS163.46 NR
H ebeloma bul biferum PR21860
H ebeloma sp. PBM2674
H ebeloma col lari atum 565
H ebeloma mesophaeum 572
H ebeloma sarcophyll um LY65BR 25
H . helodes JCS102604cc2its NR
H . helodes JCS102604B
H . helodes AWW221
H . helodes PBM2687c4
H . helodes PBM2687c2 NR
H ebeloma hel odes 688
H ebeloma cavi pes LY66BR106
H ebeloma hel odes 665
H ebeloma l utense 624
H ebeloma hi emal e LY66BR104
H ebeloma hel odes 692
H ebeloma hel odes 694
H ebeloma hel odes 651
H ebeloma hel odes 573
H ebeloma hel odes 557
H ebeloma hel odes 629
H ebeloma crustuli niforme 618
Crustuliniforme comple
H ebeloma crustuli niforme 621
H ebeloma crustuli niforme 503
H ebeloma crustuli niforme 570
H ebeloma crustuli niforme 673
H ebeloma pusi ll um 654
H ebeloma crustuli niforme 627
65
H ebeloma crustuli niforme 581
H ebeloma l utense 566
10 0
H ebeloma crustuli niforme 680
H . helodes AT2004270
H ebeloma crustuli niforme 602
H ebeloma hel odes 538
H ebeloma hel odes 539
H ebeloma pusi ll um 509
H ebeloma hel odes 650
H ebeloma hel odes 666
H ebeloma pusi ll um 645
Anami ka angustil amel la CM U
96
A. ang usti lamell a H KAS
A i ndica AF407163
Alni cola lactari olens
A al netor umAY277276
Alni cola bohemi ca 701
A amarescensAY303581
A tanti ll aAY303584
A g erani olensAY303582
A meli noides AJ296301
Alni cola escharoi des m29
A cf scol ecinaAY303583
H yal nicol a AF325632
H ytenerAF325633
H yg l acial isAF325634
H ybul li ardi AF325641
H yol ivaceus AF325642
H yg ri seus AF325636
H ypopul etor um AF325637
H yg ardner i AF325639
H ypar ksii AF325638
H ysubal pinus AF325640
H ysp T20345 AF325635
Gymnopi lus spectabi li s AFTOL
10 0
G. junoni us JC S102604A
NR
Gymnopilu
G. hybri dus JC S102604D
Ag rocybe praecox AFTOL
Ag rocybe praecox CBS108.59
x
Anamika
99
s
0.005 substituti ons/si te
Hypothesis of Hebeloma phylogeny based on ITS sequences
The above tree is a 50% majority rule consensus of 5000 neighbor joining
bootstrap replicates, adjusted with kimura-2 parameter. The monophyly of
Hebeloma is not well supported here. (Yang, in press) Taxa used in this study are
indicated.
F1
R1
relative to the cell membrane. The nitrate transporter bears the signature 12transmembrane helices and certain highly conserved sequences characteristic of
members of the Major Facilitator Superfamily. The arginine (R) residue in helix 2
(from the left) is highly conserved across several kingdoms of nitrate transporters.
Following mutation analysis in Aspergillus nidulans NRTA, it has been suggested that
this region most likely forms part of the substrate binding site. The high level of
divergence around this residue between 2 copies of the protein within an individual is
worthy of note, suggesting that function might differ between the copies.
Above: “Tanglegram” assembled in Gene Tree. On the left is a 50% majority rule concensus of 2 most
parsimonious its trees found by equally weighted branch and bound. Support values in red are posterior
probabilities of Bayesian MCMC with two hot and two cold chains for 2million generations. Support
values in black are derived from 5000 neighbor joining bootstrap replicates adjusted with kimura-2
parameter of the complete its dataset shown below and left. On the right is a neighbor joining boostrap
phylogram of inferred NRT2 amino acid sequences with 5000 replicates. Selected bootstrap values
above 70% are indicated.
Methods:
DNA was extracted from either dried fruit bodies or
rapidly growing mycelium according to a variation of the method
of____________. Sequences were PCR amplified under a variety
of conditions using 3-7 position degenerate primers designed from
a consensus of H. cylindrosporum and P. chrysosporium nrt2
sequences. ITS products were generated with ITS1F and ITS4 by
Vilgalys et al. and include ITS1, 5.8S and ITS2. Nrt2 sequences
range from 1.2-2kb.
PCR products were cloned with either TA or TOPO
TA"℠PCR 2.1 vector, selected with Xgal on LB Kanamycin agar.
10-20 clones were screened with PCR and gel electrophoresis, then
sequenced with m13 primers and 2-6 internal primers using BigDye
ver1.1. Sequences were obtained on ABI 377 and ABI3700? Data
was compiled using Sequencher"℮
Inferred intron sequences were not alignable and were
removed prior to analysis. Exon sequences were aligned manually
in MacCladeV. The large ITS alignment was first done by D.K.
Aanen, then modified by P.B. Matheny before the sequences from
this study were included.
Phylogenetic analyses were done in PAUP IV and
Mr.Bayes 3.3. Transmembrane Helical domains were confirmed by
the HMMTOP web-based algorithm.
Discussions
Phylogenetic Patterns
Assuming the tree topologies generated in these analyses are an accurate estimate of the true gene phylogenies, there are three main phenomena that require explanation.
1.The most recent common ancestor of the two nrt2 copies precedes the divergence of the Hebeloma in this dataset, and copy 2 is sister to Hebeloma.
∆This suggests that there was a duplication of the gene very early in, or preceding the emergence of Hebeloma, but after the divergence of Hebeloma and Gymnopilus. ∆ The lack of second copies of nrt2 in other
Hebeloma might suggest the loss or failure to detect many sequences, but there are potentially more plausible explanations based on the tree topologies recovered here.
2. Copy 1 of nrt2 in H. helodes is more closely allied with H.velutipes and the sweet-smelling Hebelomas than with copy 2 from the same dikaryon.
∆This is in direct conflict with the highly supported monophyletic nature of the helodes/crustuliniforme complex relative to the remaining Hebeloma in the ITS phylogeny. If we were to accept that the position of copy1
was strictly due to duplication and loss, then we would be forced to reconstruct a minimum of 24 losses based on these topologies (as calculated by GeneTree). ∆ If we broaden the possibilities of molecular evolution,
however, we might hypothesize hybridization or lateral gene transfer. In this case, we could expect the donor of copy1 to be closely related to H. sacchariolens or H. velutipes. A possible case of hybridization in the
H.velutipes clade has, in fact, been demonstrated (Aanen, 2001). ∆ Problems with these hypotheses include the likelihood of these events being of some debate, and also the lack of additional corroborative molecular
evidence such as additional ITS sequences in H. helodes. These are not necessarily fatal flaws, however, as concerted evolution in ITS or uneven crossing over during an ephemeral hybridization could explain the failure
to detect additional ITS sequences. In the case of lateral transfer, these additional factors need not be invoked, although a mechanism is required. ∆ It should be noted that H.helodes has been found associated with a
broad range of hosts, whereas potential donors of second copies have been found on only one or two hosts (Aanen, 2002); circumstantial evidence of acquisition of new ecologies through hybridization.
∆ A final explanation relies on assuming that copy1 tracks the host phylogeny with poor support, and copy2 represents a recent duplication followed by a rapid divergence due to the relaxing of constraints of selection.
Confirming this mechanism requires the uncovering of additional copy2 sequences to determine the level of similarity in the orthologs and whether there are differential rates of evolution. In this scenario, either ITS or
nrt2 (or both) is a poor indicator of host phylogeny, as they would have very different topologies.
Resolution of this issue at a minimum requires additional methods of determining nrt2 copy number in the species in this dataset.
3. The topology of the NRT2 tree appears to conflict with that of ITS along a moderately to poorly supported node, which places H.radicosum as monophyletic with H. cylindrosporum and H. edurum/sinuosum.
∆This is a result that warrants further investigation as it could suggest accelerated evolution in nrt2 along the branch to the H.radicosum terminal. H. radicosum is one of the nitrophilic species in this clade, implying that
it relies on a more reduced form of nitrogen, thus relaxing selective pressure on nrt2, in turn leading to poor bootstrap support for any clades that contain it. Future studies should include sampling the remaining
nitrophilic Hebeloma to eventually test relative rates of evolution.
∆ Different methods have suggested different topologies in NRT2 phylogeny, however both parsimony and distance analyses of amino acids suggest the topology presented above. It is possible that third base saturation
with homoplasy is responsible for alternative topologies in branch and bound of the exon nucleotide sequences. Furthermore, ITS appears to be a poor choice for resolving the backbone of the Hebeloma clade, implying
that it is a poor indicator of species phylogeny.
References:
Jargeat, Patricia et al. “Characterixation and expression
analysis of a nitrate transporter and nitrate reductase genes, two members of
a gene cluster for nitrate assimilation from the symbiotic basidiomycete
Hebeloma cylindrosporum.” Curr Genet (2003) 43: 199-205.
Marmeisse, R., et al. “Hebeloma
cylindrosporum-a model species to
study ectomycorrhizal symbiosis
from gene to ecosystem.”
New Phytologist (2004) 163: 481-498.
Aanen, Duur K., et al. “Phylogenetic
relationships in the genus Hebeloma
based on ITS1 and 2 sequences,
with special emphasis on the Hebeloma
crustuliniforme complex.” Mycologia
(2002) 92(2):269-281.
Unkles, Sheila E., et al. “Two perfectly
conserved arginine residues are required
for substrate binding in a high-affinity
Yang, et al., (in press) “New Asian species of the
genus Anamika (euagarics, hebelomatoid
clade) based on morphology and ribosomal DNA sequences.”
nitrate transporter.” PNAS (2004) 101(50): 17549-17554.
Unkles, Shelia E., et al., “Apparent genetic redundancy facilitates
ecological plasticity for nitrate transport.” The EMBO Journal (2001)
20(22): 6246-6255.
Aanen, Duur K., et al., “A widely distributed ITS polymorphism within a
biological
species of the ectomycorrhizal fungus Hebeloma velutipes.” (2001). Mycol.
Res. 105 (3): 284-290
Structural Comparison of Copies
In Aspergillus, the duplication of nrt appears to predate the divergence of A.
nidulans and A. fumigatus. It has been demonstrated that nrtA and nrtB in
Aspergillus nidulans are expressed under different concentrations of
environmental nitrate, thereby giving the species environmental “plasticity”.
Similarly, in Hebeloma, the sequence similarity between two paralogous
copies is less than that between orthologous copies in any other two species.
This could certainly be an effect of sampling, however the level of divergence
between the two copies is still noteworthy, and does not appear to be due to
the existence of a pseudogene or merely random mutation, as key functional
residues are well conserved, most substitutions are conservative, the gene
appears to be fully functional, and substitutions are clustered in specific
locations. The most notable substitutions occur in putative transmembrane
helix 2, surrounding the arginine residue named R87 (Unkles, 2004). This
residue has been found to be essential to nitrate transport and is most likely
the site of nitrate binding (Unkles, 2004). To have a high concentration of
substitutions located around R87, while R87 and its position remain intact
could suggest a retained, yet differential capacity for binding nitrate.
Aspergillus nidulans NRTB, which is suggested to be more active in high
ambient nitrate, with 1/10th the Km for binding nitrate(Unkles, 2004), shares a
common FV signature 2 residues upstream of R87 with copy1 in H. helodes
(but not copy2 or NRTA), which is more allied with the high affinity transporter
of H. cylindrosporum, a weak suggestion that copy 1 is the higher affinity
locus. There is also a high degree of divergence in the putative intracellular
loop which could be involved in regulation. It is therefore plausible that H.
helodes has acquired by hybridization or duplication, a system of nitrate
transport with variable affinity which enhances survival under fluctuating soil
nitrogen status. Under what conditions is each expressed? Do both
sequences occur in a cluster, and if so, is it the same cluster or a distant
locus? Are there additional copies of nitrate and nitrite reductase? Is there a
correlation between gene expression and mycorrhization?
NRTBAspergillusfumigatus
F3
F2 R1.5
NRTAAspergillusnidulans
NRTBAspergillusnidulans
100
Acknowledgements
I express the sincerest appreciation to Dr. Brandon
Matheny for his expertise and insight regarding basidiomycete
taxonomy
and molecular methods. Thanks also to Zhang Wang for help with
PCR troubleshooting and
Dr. Manfred Binder
for help with
phylogenetic
analysis among
other pitfalls.
Also thanks
to Lisa Bukovnik
at the sequencing
facility at Duke
University for
enabling me to
build my data set
during times of
adverse sequencing conditions.
THIS WORK WAS SUPPORTED BY NSF GRANT # DEB0228657
Below: The complete amino acid sequence of NRT2 (Jargeat et al, 2003). Inferred transmembrane helical motifs are shown as published and confirmed by HMMTOP. The putative protein kinase C
phosphorylation site is indicated. Primers --> used in this study are indicated by direction of replication. Conserved intron positions are noted by a
and F1-R2. The inferred intracellular loop is also shown.
NJ
. Most sequences represented here cover the ranges, F1-R3
R2
ASCOMYCOTA
100
ASCOMYCOTA
Phanerochaetechrysosporium
(WHITE ROTTER )
Ustilagomaydis
(PARASITE )
52
100
Coprinopsiscinerea
94
(DUNG SAPROTROPH )
100
Gymnopilusjunonius (WHITE ROTTER )
92
79
78
H.edurum
H.radicosum
H.sinuosum
H.cylindrosporum
Hhelodespbm2687copy1
Hebelomasp.jcs91904A
Hebelomasp.pbm2693
H.helodesjcs102604C
H.truncatum H.velutipes
H.helodespbm2687copy2
HEBELOMA (ECM)
BASIDIOMYCOTA
0.05changes
An hypothesis of the phylogeny of NRT2 in Fungi: The nrt2 gene phylogeny presented here
agrees with the broad, underlying phylogeny of fungal evolution, with more than one origin of
second loci suggested. Inferred amino acid sequences were used to generate similar trees with
distance and parsimony methods. This is a neighbor joining tree adjusted with Kimura-2 parameter.
Branch lengths are mean character differences. Bootstrap values were obtained by 5000 neigborjoining replicates, adjusted with Kimura-2 parameter.
R2.5
R3
MSPPKSSRGAPKFKWSHLWEPAIVNPVNLKSYTIPIFNLGDPYARAFHLSWLGFFVAFLSWFAFPPLIPDAIKSDLHLSAAQVANSNIIALCATFVVRVGVGPLVDQYGPRKVMAYLLILGAIPSGLAGTARSAEGLYVLRFFIGILGATFVPCQAWTSAFFDKNCVGTANALVGGWGNMGGGATFAIMTSLFQSLTQTYGLSTHVAWRAAFAIVPAPILLFVAVLTFIFGQDH HQIPNIQPEKSLKSSSESSKDEKDPEGNAAVTVRPAIADEDLALVKSTVDVAINEPLTLKTTVKILTNPLTWLPALAYLTTFGVELAIDSKFADVLFVLFSKRRPGFDQTTAGYYTSILGLLNLVTRPAGGYFGDLVYRHYGTNGKKAWTLLCGLIMGAALVAGGFYMQNNRTSGDEQLSVLMGVFSVAAIFSEFGNGANFALVPHCNAYNNGVMSGLVGSFGNLGGIIFAL ALLIPVSVPAL(521)
PAGKWSERHTLPVAALAVQQG
VFRFQTEVGKAFWIMGVISIGIN