Lessons from my favorite symbionts,
Questions from my favorite inscape
SAMSI Workshop on Algebraic Methods in Systems Biology and Statistics
Research Triangle Park, North Carolina14-17 September 2008
Christopher L. Schardl
Epichloë/Neotyphodium in a grass plant
• Symbioses are:– Systemic– Constitutive– Often heritable
• Symbiotic continuum:– Mutualistic– Pleiotropic– Antagonistic
Vertical transmissibility
Lolium pratense shoot and meristem with Epichloë festucae (w/GFP)
Confocal micrograph by Dr. Koya Sugawara
Lolium perenne embryo with Epichloë festucae (w/GFP)
Christensen et al. 2008
Endophytes protect against insects, nematodes, etc.
E–
E+
nutritionshelter
dispersal
anti-insectanti-vertebrateanti-nematodedrought toleranceetc.
Currencies of grass-endophyte mutualisms
Sexual vs. asexual,Mutualistic vs. antagonistic
Biological questions
• Can we elucidate patterns of host and symbiont/parasite codivergence?
• What happens (phylogenetically) during invasion of new niches?
• How do sex and asex affect evolution?• Do neofunctionalized genes have unusual
evolution ?
Hypothesis:
• Pooideae and epichloae have codiverged.
Schardl CL, Craven KD, Speakman S, Stromberg A, Lindstrom A, Yoshida R. 2008. Systematic Biology 57: 483-498.
Hosts of Epichloë spp.
Hosts
Phleum pratense, Anthoxanthum odoratumBrachypodium spp.
Poa nemoralis, Poa trivialis, Dactylis glomerata, Puccinellia distans, Lolium perenne
AveneaeBrachypodieae
Poeae E. typhina (I)
Brachypodium sylvaticumBrachypodieae E. sylvatica (VII)Holcus lanatusPoeae E. clarkii (I)
Glyceria striataMeliceaeE. glyceriae (VIII)Festuca spp., Lolium spp., Koeleria sp. Poeae, AveneaeE. festucae (II)
Elymus spp.TriticeaeE. elymi (III)
Bromus spp.BromeaeE. bromicola (VI)
Brachyelytrum erectumBrachyelytreaeE. brachyelytri (IX)Holcus mollisAveneaeEpichloë sp.
Agrostis spp., Sphenopholis spp.AveneaeE. amarillans (IV)Host tribeEpichloë sp. (MP)
Roegneria kamojiTriticeaeEpichloë yangzii (VI)
Agrostis spp., Calamagrostis spp.AveneaeE. baconii (V)
Achnatherum sibiricum
StipeaeEpichloë sp.
Epichloë gene trees
Lineage sorting effects and the species cloud
Host and epichloë phylogeniesHost cpDNA Fungus tubB + tefA
Problem with pairwise distance approach
Distancesa b c d
a 0 2 6 6b 0 6 6c 0 3d 0a b
ef
g
c d
phylogenetic tree
t = 3
Pairwise distancesto compare divergence times
Host treeA B
E F
G
C D a be f
g
c dEndophyte tree (E,e)
(F,f)(G,g)
pw d
istan
ce (E
ndop
hyte
)
pw distance (Host)MRCA pair Pairs of H and E taxon pairs(E,e) ((A,B),(a,b))(F,f) ((C,D),(c,d))(G,g)((A,C),(a,c)), ((A,D),(a,d)), ((B,C),(b,c)), ((B,D),(b,d))
MRCALink: Sample each pair of nodes once if ‘valid,’ otherwise not.
Host treeA B
E F
G
C D a be f
g
c dEndophyte tree (E,e)
(F,f)(G,g)
Node
age
(End
ophy
te)
Node age (Host)MRCA pair Pairs of H and E taxon pairs(E,e) ((A,B),(a,b))(F,f) ((C,D),(c,d))(G,g)((A,C),(a,c)), ((A,D),(a,d)), ((B,C),(b,c)), ((B,D),(b,d))
MRCALink on incongruent trees
A BE F
G
C DHost tree
a be
fg
c dEndophyte tree
MRCA pair Pairs of H and E taxon pairs(E,e) ((A,B),(a,b))(G,f)
((C,D),(c,d))(G,g) ((A,D),(a,d)), ((B,D),(b,d))(F,g)
((A,C),(a,c)), ((B,C),(b,c))
Node
age
(End
o)
Node age (Host)
(E,e)(G,f)
(G,g)(F,g)
Apply MRCALink to Pooideae-epichloae
Full
p = 0.123
Bivariate plots: Full and trimmed
p < 0.001
Codivergence of epichloae and Pooideae.• Suggests ancestral symbiosis 30–40
Mya.
Working on identifying likely host jumps
Hypothesis: E. typhina is a complex of cryptic, host-based species.
• Test for differentiation of populations based on hosts.
• Hosts sampled:– Dactylis
glomerata– Poa trivialis– Poa nemoralis– Brachypodium
pinnatum
Sites sampled in and near Switzerland
tubB haplotypes
• Support for cryptic species hypothesis.– No haplotypes
shared between host-associated populations.
• but some popns were not monophyletic
Poa trivialis
D. glomerata
D. glomerata
Poa nemoralisBp. pinnatum
H. lanatus
HOSTS:
Cautionary tale for phylogenetics
• Compatibility analysis with Carbonne’s SNAP workbench – Intron 1 shows
evidence of extensive recombination
Poa trivialis
D. glomerata
D. glomerata
Poa nemoralisBp. pinnatum
H. lanatus
HOSTS:
Remove incompatible region
1H1 Bp 12
H2 Dg 1H5 Dg 2H6 Dg 1H7 Dg 4
H9 Dg 52H12 Dg 1H14 Dg 2H15 Dg 3
H11 Dg 4H3 Dg 3
H8 Dg 10H13 Dg 1
H17 Dg 23
H16 Pn 8H18 Pn 18
H4 Bp 17H10 Bp 9
6
1
1
2
1111
111
4
2
3
11
1
1
ex D. glomerata
ex Poa nemoralis
ex Bp. pinnatum
• Looks a bit better.
Hypothesis: Recombination is associated with new colonization
events.Poa trivialis
D. glomerata
D. glomerata
Poa nemoralis
Bp. pinnatum
H. lanatus
HOSTS:
D. glomerata OREGON
D. glomerata OREGON
Sex vs. Asex
• Hypothesis: asexual lineages have shortened life spans.
tefA
C
S
C
C
CC
CC
C
C
CC
C C
S
S
S
S
S
S
S SS
SS
SS
S
S
SS
S
tubB
S
S
S
SS
S S
C
C
C
SC
CC
CS
SC
S
SS
SSS
SS
S
S
C
C
CC
C
S
clonalsexual
Sexual and clonal taxa on the gene trees
• Work of Jan Schmid & Barbara Howlett, Massey University.
tefA
C
S
C
C
CC
CC
C
C
CC
C C
S
S
S
S
S
S
S SS
SS
SS
S
S
SS
S
tubB
S
S
S
SS
S S
C
C
C
SC
CC
CS
SC
S
SS
SSS
SS
S
S
C
C
CC
C
S
clonalsexual
Sexual and clonal taxaHybrids excluded
Many asexual epichloae have multiple gene copies
• Southern blot of -tubulin genes
Genome sizes (Mb)
Neotyphodium coenophialum
57
Epichloë festucae
29
Epichloë typhina
29
Neotyphodium sp. LpTG-2
55
Kuldau et al. 1999
Hybrid origins of most asexual
epichloae
Moon et al. 2004
Phylogenetic tracking and hybridization
• Hypothesis: parasexual recombination extends life of asexual lineages.
Lolium sp.Lolium sp.
L. arundinaceumL. multiflorum
Neotyphodium sp. FaTG-3Neotyphodium sp. FaTG-2
N. coenophialumN. occultans
E. typhina
E. bromicola
E. festucae
E. typhina , E. festucae
NO
CH3
N
CHO
Hypothesis: Genes for conditionally dispensable functions have unusual
evolutionary patterns.
• Loline alkaloids
Loline biosynthesis pathway
• Novel -substitution rxn• Unusual ether bridge
Blankenship et al. 2005Faulkner et al. 2006
Relationships and proposed role of LolC
• Neofunctionalization
cysD and lolC relationships
• Why?– Paralogs with many
losses?– Long-branch
attraction?– Horizontal transfer?
lolC vs. tub2 phylogeny
• NSF • USDA-NRI• USDA-ARS
Acknowledgments• Collaborators:
– Jerzy W. Jaromczyk (UK)– Robert B. Grossman (UK)– Daniel G. Panaccione
(West Virginia Univ.) – Bruce Roe (Univ.
Oklahoma)– Barry Scott (Massey Univ.,
New Zealand)– Jan Schmid (Massey Univ.,
New Zealand) – Ruriko Yoshida (UK)– Carolyn Young (Noble
Foundation)
• UK-AGTC:– Abbe Kesterson– Jennifer Webb– & al.
• NSF • USDA-NRI• USDA-ARS
Acknowledgments• Lab:
– Kalina Andreeva– Jimmy D. Blankenship– Alfred D. Byrd– Jerome R. Faulkner– Simona Florea– Love Gill– Uljana Hesse– Walter Hollin– Eun Jung Lee– Jinge Liu– Caroline Machado
• Lab:– Lesley J. Mann– Christina D. Moon– Padmaja Nagabhyru– Kathryn Schweri– Martin J. Spiering– Huei-Fung Tsai – Jinghong Wang– Ella V. Wilson– Dong-Xiu Zhang– many undergraduate
scholars
E. typhina from D. glomerata, 2Nem
SH
ZH
VD
50 km
3.12
39.6
21.2
2.03
0.90
26.4
Q = 2Neµ
SH
ZH
VD
50 km
0.011
0.012
0.001
Acknowledgments
• Bruce Roe (UO, Norman)• Jerzy W. Jaromczyk (UK)• Wayne Beech (UK)• Mark L. Farman (UK)• Arny Stromberg (UK)
Funding:• NSF • USDA-NRI• USDA-ARS
• Uljana Hesse• Kalina Andreeva• Dongxiu Zhang• Ellie Arnaoudova• Paul Maynard• Na Ren• Venu-Gopal Puram• Jennifer L. Wiseman• Jennifer Webb• Abbe Kesterson• Love Gill• S. Macmil• G. Wiley
Loline biosynthesis pathway
• Novel -substitution rxn– Probably catalyzed by
LolC• Unusual ether bridge
Blankenship et al. 2005Faulkner et al. 2006
tubB tefA
ETC ETC
Epichloë phylogeny
Lolines protect against insects
• Plants with lolines are resistant to bird-cherry oat aphid (Rhopalosiphum padi)
0
20
40
60livedead
Num
ber
of A
phid
s
E. festucaeLol+ Lol–
67–576
ba c
E– Nun
367–4871
Wilkinson et al. 2000
cysD and lolC relationships
• Why?
Polymorphisms for alkaloid expression
Species Host Lolines Ergovaline
Epichloë amarillans Agrostis hiemalis + -E. amarillans A. perennans - -Epichloë festucae Festuca rubra - +E. festucae Lolium giganteum + -
Genetic identification of LOL locus• 1:1 Segregation
– Implies single gene locus• (These fungi are
haploids)
Wilkinson et al. 2000
Loline biosynthesis gene cluster
Spiering et al. 2005
PLPbinding
Hemebinding
Myb DNAbinding
PLPbinding
HxD...HFacialtriad
PLPbinding
FADbinding
HxD...HFacialtriad
0 5 10 15 20 25 kb
lolC lolD lolO lolA lolU lolP lolT lolElolF
• The LOL1 gene cluster from Neotyphodium uncinatum
Lol protein relationshipsGene Closest BLAST or CD match Putative Rxn TypelolE Epoxidase Nonheme iron oxygenaselolT Isopenicillin N epimerase Pyridoxal phosphate (PLP)lolP Pisatin demethylase P450 monooxygenaselolU Myb transcription factor DNA bindinglolA Asp kinase (allosteric domain only) Amino acid bindinglolO Thymine 7-hydroxylase Nonheme iron oxygenaselolD Ornithine decarboxylase PLPlolC O-Acetylhomoserine (thiol) lyase PLPlolF 1,2-Cyclopentanone monoox. FAD monooxygenase
Spiering et al. 2005
Ergot alkaloids
LSD
NH2C
CH3
CH2
CH3
CO N
CH3
H
NH
H
Drunken horse grass
St. Anthony’s Fire Salem witch trials
Fescue toxicosis
Medicinal uses for Childbirth, Migraines, Parkinsonism
Ergot alkaloid biosynthesis gene clusters in Claviceps spp.
C. fusiformis
C. purpurea
lpsB
easA
cloA easD
easEeasC easG
easF dmaW
Clavines
Ergopeptines
easEeasC easG
cloA easD easF dmaW
lpsB
easA
easH lpsA1 lpsA2easHlpsC
Haarmann &al. 2005Caroline MachadoElla Wilson
dmaW encodes dimethylallylTrp synthase
GENUS FUNCTION E-VALUE IN CLUSTER?
Epichloë DMATrp synthase 0.0 Yes
Balansia DMATrp synthase 4e-170 ?Claviceps DMATrp synthase 6e-170 YesAspergillus DMATrp synthase 2e-156 YesAspergillus Reverse prenylation 9e-41 YesNeurospora Hypothetical protein 6e-32 ?Magnaporthe Hypothetical protein 6e-32 YesAspergillus Terrequinone biosyn. 7e-21 YesFusarium Equisetin biosyn.? 1e-17 ?Leptosphaeria Sirodesmin biosyn. 6e-16 YesPenicillium Hypothetical protein 1e-7 Yes
DmaW BLAST matches
DmaW in clavicipitaceous symbionts of Convolvulaceae
• Phylogeny of DmaW family prenyltransferases:
Steiner &al. 2006
Unsöld & Li, Chembiochem (2006)
Reverse prenylation in Aspergillus fumigatus
Terrequinone A in Aspergillus
nidulans (Bok &al. 2006)
Roquefortine in Penicillium
roquefortii (Steiner &al 2006)
Other prenylated fungal metabolites
Conclusions
• Long term Pooideae-epichloë codivergence• Extensive hybridization in endophyte evolution• Secondary metabolism genes
– Clustering– Neofunctionalization– Presence/absence polymorphisms
• Gene expression differences– Benign vs pathogenic expression
p < 0.001 p < 0.001
Working on identifying likely host jumps
Ergot alkaloid biosynthesis pathway
Floss 2006Schardl &al. 2006
Lolines protect against insects
• Plants with lolines are resistant to bird-cherry oat aphid (Rhopalosiphum padi)
0
20
40
60livedead
Num
ber
of A
phid
s
E. festucaeLol+ Lol–
67–576
ba c
E– Nun
367–4871
Wilkinson et al. 2000
Life cycles of Epichloë and Neotyphodium spp.
asexual cycle & vertical
transmission
sexual cycle & horizontal transmission
Full
p = 0.123
p < 0.001
Bivariate plots: Full and ETC trimmed