Transcript Document

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 .edu ru mC BS291 .50 NR
H . sin uo sum C BS1 84 .47
H ebe loma e du rum 63 7 NR
H ebe loma d an icu mL Y64 BR 38
60
H ebe loma c ylin dr osp oru m 610 0
H .cyl ind ros por um CBS5 58.96 NR
H ebe loma b irru s 5 80
H ebe loma r adicos um 64 0
nitrophili c
H .radicosumC BS183 .47 NR
H ebe loma c iric na ns 6 38
H ebe loma s ina piz ans 51 4
H ebe loma trun catum 641
H ebe loma s p.PBM269 3c 3
H ebe loma s p.PBM269 3c 2 NR
H ebe loma s p. J CS91 904 A NR
H ebe loma s p. PBM26 91
59
H ebe loma s ac cha rio len s 5 52
H ebe loma tome nto su m5 06
H . tru nca tum C BS2 95 .50
NR
2
H ebe loma v elu tipe s 5 04 1
H ebe loma v elu tipe s 5 35
H ebe loma v elu tipe s 6 42
H ebe loma i nca rna tulu m 527
H ebe loma v elu tipe s 5 40
H . ve lutip es UP18 1
H.v elutipes clad
e
H ebe loma v elu tipe s AFTOL
H ebe loma v elu tipe s 5 02
H ebe loma v elu tipe s 5 04 2
86
H .vel utip es CBS16 3.4 6 NR
H ebe loma b ulb ifer um PR 21 860
H ebe loma s p. PBM26 74
H ebe loma c olla ria tum 56 5
H ebe loma mes op hae um 57 2
H ebe loma s arc op hyl lum LY65BR2 5
H . he lod es JCS10 260 4c c2 its NR
H . he lod es JCS10 260 4B
H . he lod es AWW2 21
H . he lod es PBM26 87c 4
H . he lod es PBM26 87c 2 NR
H ebe loma h elo de s 68 8
H ebe loma c av ipe s LY66 BR 106
H ebe loma h elo de s 66 5
H ebe loma l uten se 62 4
H ebe loma h iemale LY66 BR 104
H ebe loma h elo de s 69 2
H ebe loma h elo de s 69 4
H ebe loma h elo de s 65 1
H ebe loma h elo de s 57 3
H ebe loma h elo de s 55 7
H ebe loma h elo de s 62 9
H ebe loma c rus tul inifo rme 6 18
Crustuliniformecomple
H ebe loma c rus tul inifo rme 6 21
H ebe loma c rus tul inifo rme 5 03
H ebe loma c rus tul inifo rme 5 70
H ebe loma c rus tul inifo rme 6 73
H ebe loma p us illum 65 4
H ebe loma c rus tul inifo rme 6 27
65
H ebe loma c rus tul inifo rme 5 81
H ebe loma l uten se 56 6
10 0
H ebe loma c rus tul inifo rme 6 80
H . he lod es AT2 00 427 0
H ebe loma c rus tul inifo rme 6 02
H ebe loma h elo de s 53 8
H ebe loma h elo de s 53 9
H ebe loma p us illum 50 9
H ebe loma h elo de s 65 0
H ebe loma h elo de s 66 6
H ebe loma p us illum 64 5
An amika an gus tila me lla C MU
96
A. ang us tila mel la H KAS
A indi ca AF4 07 163
Al nico la lac tario len s
A alne tor umAY2 77 276
Al nico la boh emica 70 1
A ama re sce ns AY3 03 581
A tantillaAY3 035 84
A ger ani ole nsAY3 035 82
A mel ino ide s AJ 29 630 1
Al nico la esc ha roid es m2 9
A cf s col eci naAY3 035 83
H y al nic ola AF3 25 632
H y te ner AF325 63 3
H y gl aci alis AF3 25 634
H y bu llia rdi AF3 25 641
H y ol iva ceu s AF3 256 42
H y gr ise us AF3 25 636
H y po pu leto rum AF32 56 37
H y ga rd ner i AF32 563 9
H y pa rk sii AF3 256 38
H y su ba lpin us AF325 64 0
H y sp T2 03 45 AF3 25 635
Gymn op ilus sp ec tabi lis AFTOL
10 0
G. jun on ius JC S10 26 04A NR
Gymnopilu
G. hy brid us JC S1 026 04 D
Ag roc yb e p rae co x AFTOL
Ag roc yb e p rae co x C BS1 08 .59
x
Anamika
99
s
0.005 su bs tituti ons /site
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
ASCOMYCOTA
100
Phanerochaetechrysosporium
(WHITE ROTTER )
Ustilagomaydis
(PARASITE )
52
100
Coprinopsiscinerea
94
(DUNG SAPROTROPH )
Gymnopilusjunonius (WHITE ROTTER )
100
92
79
H.edurum
H.radicosum 78
H.sinuosum
H.cylindrosporum
Hhelodespbm2687copy1
Hebelomasp.jcs91904A
Hebelomasp.pbm2693
H.helodesjcs102604C
H.truncatum H.velutipes
H.helodespbm2687copy2
HEBELOMA (ECM)
BASIDIOMYCOT A
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