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