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Lessons from my favorite symbionts , Questions from my favorite inscape . Christopher L. Schardl. SAMSI Workshop on Algebraic Methods in Systems Biology and Statistics Research Triangle Park, North Carolina 14-17 September 2008. Epichloë/Neotyphodium in a grass plant. Symbioses are:

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lessons from my favorite symbionts questions from my favorite inscape

Lessons from my favorite symbionts,Questions from my favorite inscape

Christopher L. Schardl

SAMSI Workshop on Algebraic Methods in Systems Biology and Statistics

Research Triangle Park, North Carolina

14-17 September 2008

epichlo neotyphodium in a grass plant
Epichloë/Neotyphodium in a grass plant
  • Symbioses are:
    • Systemic
    • Constitutive
    • Often heritable
  • Symbiotic continuum:
    • Mutualistic
    • Pleiotropic
    • Antagonistic
vertical transmissibility
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
currencies of grass endophyte mutualisms
Currencies of grass-endophyte mutualisms

anti-insect

anti-vertebrate

anti-nematode

drought tolerance

etc.

nutrition

shelter

dispersal

biological questions
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
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 epichlo spp

E. amarillans (IV)

Aveneae

Agrostis spp., Sphenopholis spp.

E. baconii (V)

Aveneae

Agrostis spp., Calamagrostis spp.

Epichloë sp.

Aveneae

Holcus mollis

E. brachyelytri (IX)

Brachyelytreae

Brachyelytrum erectum

Epichloë sp.

Stipeae

Achnatherum sibiricum

E. bromicola (VI)

Bromeae

Bromus spp.

Triticeae

Epichloë yangzii (VI)

Roegneria kamoji

E. elymi (III)

Triticeae

Elymus spp.

E. festucae (II)

Poeae,Aveneae

Festucaspp., Loliumspp.,Koeleria sp.

E. glyceriae (VIII)

Meliceae

Glyceria striata

Poeae

E. clarkii (I)

Holcus lanatus

Brachypodieae

E. sylvatica (VII)

Brachypodium sylvaticum

Aveneae

Brachypodieae

Poeae

Phleum pratense, Anthoxanthum odoratum

Brachypodiumspp.

Poa nemoralis, Poa trivialis, Dactylis glomerata, Puccinellia distans, Lolium perenne

E. typhina (I)

Hosts of Epichloë spp.

Epichloë sp. (MP)

Host tribe

Hosts

host and epichlo phylogenies
Host and epichloë phylogenies

Host cpDNA

Fungus tubB + tefA

problem with pairwise distance approach

g

t = 3

f

e

a

b

c

d

phylogenetic tree

Problem with pairwise distance approach
pairwise distances to compare divergence times

(G,g)

G

g

(F,f)

f

(E,e)

E

F

e

pw distance (Endophyte)

A

B

C

D

a

b

c

d

Host tree

Endophyte tree

MRCA pair

Pairs of H and E taxon pairs

pw distance (Host)

(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))

Pairwise distancesto compare divergence times
mrcalink sample each pair of nodes once if valid otherwise not

G

g

(G,g)

f

E

F

e

Node age (Endophyte)

(F,f)

A

B

C

D

a

b

c

d

(E,e)

Host tree

Endophyte tree

MRCA pair

Pairs of H and E taxon pairs

Node age (Host)

(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.
mrcalink on incongruent trees

(F,g)

(G,g)

Node age (Endo)

(G,f)

G

g

(E,e)

f

E

F

e

Node age (Host)

A

B

C

D

a

b

c

d

MRCA pair

Pairs of H and E taxon pairs

Host tree

Endophyte tree

(E,e)

((A,B),(a,b))

(G,f)

((A,C),(a,c)), ((B,C),(b,c))

(G,g)

((A,D),(a,d)), ((B,D),(b,d))

(F,g)

((C,D),(c,d))

MRCALink on incongruent trees
codivergence of epichloae and pooideae
Codivergence of epichloae and Pooideae.
  • Suggests ancestral symbiosis 30–40 Mya.
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

Hypothesis: E. typhina is a complex of cryptic, host-based species.
tubb haplotypes

HOSTS:

Poa trivialis

D. glomerata

H. lanatus

D. glomerata

Poa nemoralis

Bp. pinnatum

tubB haplotypes
  • Support for cryptic species hypothesis.
    • No haplotypes shared between host-associated populations.
  • but some popns were not monophyletic
cautionary tale for phylogenetics

HOSTS:

Poa trivialis

D. glomerata

H. lanatus

D. glomerata

Poa nemoralis

Bp. pinnatum

Cautionary tale for phylogenetics
  • Compatibility analysis with Carbonne’s SNAP workbench
    • Intron 1 shows evidence of extensive recombination
remove incompatible region

1

H2 Dg 1

1

H5 Dg 2

1

H6 Dg 1

1

H7 Dg 4

2

H9 Dg 52

1

H12 Dg 1

1

1

ex D. glomerata

H14 Dg 2

1

H15 Dg 3

4

1

H11 Dg 4

3

H3 Dg 3

1

H8 Dg 10

2

1

H13 Dg 1

H17 Dg 23

H18 Pn 18

ex Poa nemoralis

1

H16 Pn 8

1

H1 Bp 12

ex Bp. pinnatum

1

H4 Bp 17

6

H10 Bp 9

Remove incompatible region
  • Looks a bit better.
hypothesis recombination is associated with new colonization events
Hypothesis: Recombination is associated with new colonization events.

HOSTS:

Poa trivialis

D. glomerata OREGON

D. glomerata

H. lanatus

D. glomerata

Poa nemoralis

D. glomerata OREGON

Bp. pinnatum

sex vs asex
Sex vs. Asex
  • Hypothesis: asexual lineages have shortened life spans.
sexual and clonal taxa on the gene trees
Sexual and clonal taxa on the gene trees
  • Work of Jan Schmid & Barbara Howlett, Massey University.

tefA

tubB

S

C

C

C

C

C

S

S

C

S

S

S

S

S

S

S

S

S

S

S

S

C

C

S

S

C

S

S

S

S

S

S

S

C

C

C

S

S

C

C

S

S

C

C

S

S

C

C

S

S

S

S

C

S

S

C

C

S

C

S

C

C

C

S

clonal

sexual

C

S

sexual and clonal taxa
Sexual and clonal taxa

Hybrids excluded

tefA

tubB

S

C

C

C

C

C

S

S

C

S

S

S

S

S

S

S

S

S

S

S

S

C

C

S

S

C

S

S

S

S

S

S

S

C

C

C

S

S

C

C

S

S

C

C

S

S

C

C

S

S

S

S

C

S

S

C

C

S

C

S

C

C

C

S

clonal

sexual

C

S

many asexual epichloae have multiple gene copies

Neotyphodium sp. LpTG-2

Neotyphodium coenophialum

Epichloë festucae

Epichloë typhina

55

57

29

29

Manyasexual epichloae have multiple gene copies
  • Southern blot of -tubulin genes

Genome sizes (Mb)

Kuldau et al. 1999

phylogenetic tracking and hybridization
Phylogenetic tracking and hybridization

E. bromicola

  • Hypothesis: parasexual recombination extends life of asexual lineages.

N. occultans

L. multiflorum

N. coenophialum

L. arundinaceum

E. typhina , E. festucae

E. festucae

Neotyphodium sp. FaTG-2

Lolium sp.

Lolium sp.

Neotyphodium sp.FaTG-3

E. typhina

hypothesis genes for conditionally dispensable functions have unusual evolutionary patterns

O

CH

N

CH3

O

N

Hypothesis: Genes for conditionally dispensable functions have unusual evolutionary patterns.
  • Loline alkaloids
loline biosynthesis pathway
Loline biosynthesis pathway
  • Novel -substitution rxn
  • Unusual ether bridge

Blankenship et al. 2005

Faulkner et al. 2006

cysd and lolc relationships
cysD and lolC relationships
  • Why?
    • Paralogs with many losses?
    • Long-branch attraction?
    • Horizontal transfer?
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)

RurikoYoshida (UK)

Carolyn Young (Noble Foundation)

UK-AGTC:

AbbeKesterson

Jennifer Webb

& al.

Acknowledgments
  • NSF
  • USDA-NRI
  • USDA-ARS
acknowledgments1
Lab:

KalinaAndreeva

Jimmy D. Blankenship

Alfred D. Byrd

Jerome R. Faulkner

SimonaFlorea

Love Gill

UljanaHesse

Walter Hollin

Eun Jung Lee

Jinge Liu

Caroline Machado

Lab:

Lesley J. Mann

Christina D. Moon

PadmajaNagabhyru

Kathryn Schweri

Martin J. Spiering

Huei-Fung Tsai

Jinghong Wang

Ella V. Wilson

Dong-Xiu Zhang

many undergraduate scholars

Acknowledgments
  • NSF
  • USDA-NRI
  • USDA-ARS
e typhina from d glomerata 2n e m
E. typhina from D. glomerata, 2Nem

2.03

3.12

SH

ZH

39.6

26.4

0.90

21.2

VD

50 km

q 2n e
Q = 2Neµ

0.011

SH

0.012

ZH

VD

0.001

50 km

acknowledgments2
Bruce Roe (UO, Norman)

Jerzy W. Jaromczyk (UK)

Wayne Beech (UK)

Mark L. Farman (UK)

Arny Stromberg (UK)

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

  • Funding:
  • NSF
  • USDA-NRI
  • USDA-ARS
Acknowledgments
loline biosynthesis pathway1
Loline biosynthesis pathway
  • Novel -substitution rxn
    • Probably catalyzed by LolC
  • Unusual ether bridge

Blankenship et al. 2005

Faulkner et al. 2006

lolines protect against insects

c

a

b

live

60

dead

40

367–

4871

67–

576

Number of Aphids

20

0

E–

Nun

Lol+

Lol–

E. festucae

Lolines protect against insects
  • Plants with lolines are resistant to bird-cherry oat aphid (Rhopalosiphum padi)

Wilkinson et al. 2000

genetic identification of lol locus
Genetic identification of LOL locus
  • 1:1 Segregation
    • Implies single gene locus
      • (These fungi are haploids)

Wilkinson et al. 2000

loline biosynthesis gene cluster

lolF

lolC

lolD

lolO

lolA

lolU

lolP

lolT

lolE

Myb DNA

binding

HxD...H

Facial

triad

HxD...H

Facial

triad

Heme

binding

FAD

binding

PLP

binding

PLP

binding

PLP

binding

0

5

10

15

20

25 kb

Loline biosynthesis gene cluster
  • The LOL1 gene cluster from Neotyphodium uncinatum

Spiering et al. 2005

lol protein relationships
Lol protein relationships

Spiering et al. 2005

ergot alkaloids

C

H

C

H

3

3

H

C

C

H

2

2

Drunken horse grass

C

H

3

Fescue toxicosis

St. Anthony’s Fire

N

C

N

H

O

N

H

H

LSD

Salem witch trials

Ergot alkaloids

Medicinal uses for Childbirth, Migraines, Parkinsonism

ergot alkaloid biosynthesis gene clusters in claviceps spp

easH

lpsA1

easH

lpsA2

easC

easE

easG

lpsC

lpsB

easA

cloA

easD

easF

dmaW

Ergot alkaloid biosynthesis gene clusters in Claviceps spp.

lpsB

cloA

easD

easF

dmaW

easA

easC

easE

easG

Clavines

C. fusiformis

C. purpurea

Haarmann &al. 2005

Caroline Machado

Ella Wilson

Ergopeptines

dmaw in clavicipitaceous symbionts of convolvulaceae
DmaW in clavicipitaceous symbionts of Convolvulaceae
  • Phylogeny of DmaW family prenyltransferases:

Steiner &al. 2006

reverse prenylation in aspergillus fumigatus
Reverse prenylation in Aspergillus fumigatus

Unsöld & Li, Chembiochem (2006)

other prenylated fungal metabolites
Other prenylated fungal metabolites

Terrequinone A in Aspergillus nidulans(Bok &al. 2006)

Roquefortine in Penicillium roquefortii (Steiner &al 2006)

conclusions
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
ergot alkaloid biosynthesis pathway
Ergot alkaloid biosynthesis pathway

Floss 2006

Schardl &al. 2006

lolines protect against insects1

c

a

b

live

60

dead

40

367–

4871

67–

576

Number of Aphids

20

0

E–

Nun

Lol+

Lol–

E. festucae

Lolines protect against insects
  • Plants with lolines are resistant to bird-cherry oat aphid (Rhopalosiphum padi)

Wilkinson et al. 2000

life cycles of epichlo and neotyphodium spp
Life cycles of Epichloë and Neotyphodium spp.

asexual cycle & vertical transmission

sexual cycle & horizontal transmission