lessons from my favorite symbionts , questions from my favorite inscape

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symbionts, Questions from my favorite inscape SAMSI Workshop on Algebraic Methods in Systems Biology and Statistics Research Triangle Park, North Carolina 14-17 September 2008 Christopher L. Schardl

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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: - PowerPoint PPT Presentation

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Endophyte biology and evolution

Lessons from my favorite symbionts,Questions from my favorite inscape SAMSI Workshop on Algebraic Methods in Systems Biology and StatisticsResearch Triangle Park, North Carolina14-17 September 2008Christopher L. Schardl

1Epichlo/Neotyphodium in a grass plant

Symbioses are:SystemicConstitutiveOften heritableSymbiotic continuum:MutualisticPleiotropicAntagonistic2Vertical transmissibilityLolium pratense shoot and meristem with Epichlo festucae (w/GFP) Confocal micrograph by Dr. Koya Sugawara

Lolium perenne embryo with Epichlo festucae (w/GFP)Christensen et al. 2008

3Endophytes protect against insects, nematodes, etc.

EE+

nutritionshelterdispersalanti-insectanti-vertebrateanti-nematodedrought toleranceetc.Currencies of grass-endophyte mutualisms5

Sexual vs. asexual,Mutualistic vs. antagonistic6Biological questionsCan 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: 483-498.8Hosts of Epichlo spp.HostsPhleum pratense, Anthoxanthum odoratumBrachypodium spp.Poa nemoralis, Poa trivialis, Dactylis glomerata, Puccinellia distans, Lolium perenneAveneaeBrachypodieaePoeae 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 mollisAveneaeEpichlo sp.Agrostis spp., Sphenopholis spp.AveneaeE. amarillans (IV)Host tribeEpichlo sp. (MP)Roegneria kamojiTriticeaeEpichlo yangzii (VI)Agrostis spp., Calamagrostis spp.AveneaeE. baconii (V)Achnatherum sibiricumStipeaeEpichlo sp. 9Epichlo gene treesLineage sorting effects and the species cloudHost and epichlo phylogeniesHost cpDNAFungus tubB + tefA12Problem with pairwise distance approachDistancesabcda0266b066c03d0abefgcdphylogenetic treet = 313Pairwise distancesto compare divergence times Host treeABEFGCDabefgcdEndophyte tree(E,e)(F,f)(G,g)pw distance (Endophyte)pw distance (Host)MRCA pairPairs 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))14MRCALink: Sample each pair of nodes once if valid, otherwise not. Host treeABEFGCDabefgcdEndophyte tree(E,e)(F,f)(G,g)Node age (Endophyte)Node age (Host)MRCA pairPairs 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))15MRCALink on incongruent treesABEFGCDHost treeabefgcdEndophyte treeMRCA pairPairs 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 (Endo)Node age (Host)(E,e)(G,f)(G,g)(F,g)16Apply MRCALink to Pooideae-epichloae17Fullp = 0.123Bivariate plots: Full and trimmed p < 0.00118Codivergence of epichloae and Pooideae.Suggests ancestral symbiosis 3040 Mya.19Working on identifying likely host jumps20Hypothesis: E. typhina is a complex of cryptic, host-based species.Test for differentiation of populations based on hosts.Hosts sampled:Dactylis glomerataPoa trivialisPoa nemoralisBrachypodium pinnatumSites sampled in and near Switzerland

21tubB haplotypesSupport for cryptic species hypothesis.No haplotypes shared between host-associated populations.but some popns were not monophyletic

Poa trivialisD. glomerataD. glomerataPoa nemoralisBp. pinnatumH. lanatusHOSTS:22Cautionary tale for phylogeneticsCompatibility analysis with Carbonnes SNAP workbench Intron 1 shows evidence of extensive recombination

Poa trivialisD. glomerataD. glomerataPoa nemoralisBp. pinnatumH. lanatusHOSTS:23Remove incompatible region1H1 Bp 12H2 Dg 1H5 Dg 2H6 Dg 1H7 Dg 4H9 Dg 52H12 Dg 1H14 Dg 2H15 Dg 3H11 Dg 4H3 Dg 3H8 Dg 10H13 Dg 1H17 Dg 23H16 Pn 8H18 Pn 18H4 Bp 17H10 Bp 9611211111114231111ex D. glomerataex Poa nemoralisex Bp. pinnatumLooks a bit better.24Hypothesis: Recombination is associated with new colonization events.

Poa trivialisD. glomerataD. glomerataPoa nemoralisBp. pinnatumH. lanatusHOSTS:D. glomerata OREGOND. glomerata OREGON25

Sex vs. AsexHypothesis: asexual lineages have shortened life spans.26cDNA from lab culture

tefACSCCCCCCCCCCCCSSSSSSSSSSSSSSSSSS

tubBSSSSSSSCCCSCCCCSSCSSSSSSSSSSCCCCCSclonalsexualSexual and clonal taxa on the gene treesWork of Jan Schmid & Barbara Howlett, Massey University.27cDNA from lab culture

tefACSCCCCCCCCCCCCSSSSSSSSSSSSSSSSSS

tubBSSSSSSSCCCSCCCCSSCSSSSSSSSSSCCCCCSclonalsexualSexual and clonal taxaHybrids excluded28cDNA from lab cultureMany asexual epichloae have multiple gene copiesSouthern blot of -tubulin genesGenome sizes (Mb)Neotyphodium coenophialum57Epichlo festucae29Epichlo typhina29Neotyphodium sp. LpTG-255Kuldau et al. 199929Hybrid origins of most asexual epichloae

Moon et al. 200430Phylogenetic tracking and hybridizationHypothesis: parasexual recombination extends life of asexual lineages.

Lolium sp.Lolium sp.L. arundinaceumL. multiflorumNeotyphodium sp. FaTG-3Neotyphodium sp. FaTG-2N. coenophialumN. occultansE. typhinaE. bromicolaE. festucaeE. typhina , E. festucae31NOCH3NCHOHypothesis: Genes for conditionally dispensable functions have unusual evolutionary patterns.Loline alkaloids32Loline biosynthesis pathwayNovel -substitution rxnUnusual ether bridgeBlankenship et al. 2005Faulkner et al. 200633Relationships and proposed role of LolCNeofunctionalization34cysD and lolC relationshipsWhy?Paralogs with many losses?Long-branch attraction?Horizontal transfer?lolC vs. tub2 phylogeny

36NSF USDA-NRIUSDA-ARS

AcknowledgmentsCollaborators: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 KestersonJennifer Webb& al.

37NSF USDA-NRIUSDA-ARS

AcknowledgmentsLab:Kalina AndreevaJimmy D. BlankenshipAlfred D. ByrdJerome R. FaulknerSimona FloreaLove GillUljana HesseWalter HollinEun Jung LeeJinge LiuCaroline MachadoLab:Lesley J. MannChristina D. MoonPadmaja NagabhyruKathryn SchweriMartin J. SpieringHuei-Fung Tsai Jinghong WangElla V. WilsonDong-Xiu Zhangmany undergraduate scholars

38

E. typhina from D. glomerata, 2NemSHZHVD50 km3.1239.621.22.030.9026.440

Q = 2NeSHZHVD50 km0.0110.0120.00141AcknowledgmentsBruce Roe (UO, Norman)Jerzy W. Jaromczyk (UK)Wayne Beech (UK)Mark L. Farman (UK)Arny Stromberg (UK) Funding:NSF USDA-NRIUSDA-ARS

Uljana HesseKalina AndreevaDongxiu ZhangEllie ArnaoudovaPaul MaynardNa RenVenu-Gopal PuramJennifer L. WisemanJennifer WebbAbbe KestersonLove GillS. MacmilG. Wiley

42Loline biosynthesis pathwayNovel -substitution rxnProbably catalyzed by LolCUnusual ether bridgeBlankenship et al. 2005Faulkner et al. 200643tubBtefA

ETCETCEpichlo phylogeny44Lolines protect against insectsPlants with lolines are resistant to bird-cherry oat aphid (Rhopalosiphum padi)0204060livedeadNumber of AphidsE. festucaeLol+Lol67576

bacENun3674871Wilkinson et al. 2000cysD and lolC relationshipsWhy?Polymorphisms for alkaloid expressionSpeciesHostLolinesErgovalineEpichlo amarillansAgrostis hiemalis+-E. amarillansA. perennans--Epichlo festucaeFestuca rubra-+E. festucaeLolium giganteum+-48Genetic identification of LOL locus1:1 SegregationImplies single gene locus(These fungi are haploids)Wilkinson et al. 2000

49Loline biosynthesis gene clusterSpiering et al. 2005PLPbindingHemebindingMyb DNAbindingPLPbindingHxD...HFacialtriadPLPbindingFADbindingHxD...HFacialtriad0510152025 kb

lolClolDlolOlolAlolUlolPlolTlolElolFThe LOL1 gene cluster from Neotyphodium uncinatum50Figure 8. Diagram of LOL clusters in N. uncinatum. Hashed horizontal lines indicate the sequenced regions. Shaded rectangles indicate coding sequences, and white rectangles indicate introns. Long vertical lines indicate signature sequences for PLP binding sites in LolC, LolD and LolT, an FAD binding site in LolF, and a Myb DNA binding site in LolU. Arrows indicate directions and minimum lengths of transcripts. No single long open reading frame was predicted from the transcribed seqX sequence. The lolP copy in LOL2 has a deletion that truncates the open reading frame. Data are from Spiering et al. 2005.Lol protein relationshipsGeneClosest BLAST or CD matchPutative Rxn TypelolEEpoxidaseNonheme iron oxygenaselolTIsopenicillin N epimerasePyridoxal phosphate (PLP)lolPPisatin demethylaseP450 monooxygenaselolUMyb transcription factorDNA bindinglolAAsp kinase (allosteric domain only)Amino acid bindinglolOThymine 7-hydroxylaseNonheme iron oxygenaselolDOrnithine decarboxylasePLPlolCO-Acetylhomoserine (thiol) lyasePLPlolF1,2-Cyclopentanone monoox.FAD monooxygenaseSpiering et al. 200551Ergot alkaloidsLSDNH2CCH3CH2CH3CONCH3HNHH

Drunken horse grass

St. Anthonys Fire

Salem witch trials

Fescue toxicosisMedicinal uses for Childbirth, Migraines, Parkinsonism52Ergot alkaloid biosynthesis gene clusters in Claviceps spp.C. fusiformisC. purpurealpsBeasAcloAeasDeasEeasCeasGeasFdmaWClavinesErgopeptineseasEeasCeasGcloAeasDeasFdmaWlpsBeasAeasHlpsA1lpsA2easHlpsCHaarmann &al. 2005Caroline MachadoElla Wilson53dmaW encodes dimethylallylTrp synthase54GENUSFUNCTIONE-VALUEIN CLUSTER?EpichloDMATrp synthase0.0YesBalansiaDMATrp synthase4e-170?ClavicepsDMATrp synthase6e-170YesAspergillusDMATrp synthase2e-156YesAspergillusReverse prenylation9e-41YesNeurosporaHypothetical protein6e-32?MagnaportheHypothetical protein6e-32YesAspergillusTerrequinone biosyn.7e-21YesFusariumEquisetin biosyn.?1e-17?LeptosphaeriaSirodesmin biosyn.6e-16YesPenicilliumHypothetical protein1e-7YesDmaW BLAST matches55

DmaW in clavicipitaceous symbionts of ConvolvulaceaePhylogeny of DmaW family prenyltransferases:Steiner &al. 2006Unsld & Li, Chembiochem (2006)Reverse prenylation in Aspergillus fumigatusTerrequinone A in Aspergillus nidulans (Bok &al. 2006)Roquefortine in Penicillium roquefortii (Steiner &al 2006)Other prenylated fungal metabolitesConclusionsLong term Pooideae-epichlo codivergenceExtensive hybridization in endophyte evolutionSecondary metabolism genesClusteringNeofunctionalizationPresence/absence polymorphismsGene expression differencesBenign vs pathogenic expression p < 0.001 p < 0.001Working on identifying likely host jumps60Ergot alkaloid biosynthesis pathwayFloss 2006Schardl &al. 200661Lolines protect against insectsPlants with lolines are resistant to bird-cherry oat aphid (Rhopalosiphum padi)0204060livedeadNumber of AphidsE. festucaeLol+Lol67576

bacENun3674871Wilkinson et al. 2000

Life cycles of Epichlo and Neotyphodium spp.asexual cycle & vertical transmissionsexual cycle & horizontal transmission

63Fullp = 0.123 p < 0.001Bivariate plots: Full and ETC trimmed64

NH

O O

O

O

O

O

OH

NO

NR2

R1

NN

NH

HN

NH2

O

CH3

HNN

CO H

N O

NN

O O

HO

tefA

0.005 substitutions/site

tubB

b

MRCASMRCAS

1 2 now

MRCAG(1,2)

3 4

A

now

MRCAG(1,2)

1 2 3 4

B

1 2 3' 3 4

D

now

MRCAG(3',4)

MRCAG(3,4)

1 2 3' 3 4

C

now

MRCAG(3',4)

MRCAG(3,4)

Brachyelytrum erectumAchnatherum inebriansGlyceria striataElymus canadensis

Hordeum brevisubulatumHordelymus eurupaeusBromus ramosusBromus erectusBromus purgansLolium sp. P4078Lolium sp. P4074Lolium arundinaceumLolium multiflorumLolium edwardiiLolium perenneFestuca rubraFestuca longifolia

Holcus mollisAgrostis teniusAgrostis hiemalisCalamagrostis villosaEchinopogon ovatusKoeleria cristataSphenopholis obtusataBrachypodium sylvaticum

0.01 substitutions/site 0.01 substitutions/site

N. lolli

E. brachyelytriN. gansuenseE. glyceriae

N. sp. HbTG1

E. bromicolaE. bromicola

N. sp. HeTG1

E. elymiE. elymi

N. sp. FaTG2N. sp. FaTG3N. coenophialumN. occultansE. baconii

E. festucaeE. festucaeE. festucaeEpichlo sp. E. amarillansE. amarillansE. baconiiN. aotearoaeN. typhinumE. typhinaE. sylvatica

0.98

0.99

0.97

0.620.63

1

1

1

1

11

1

1

1

1

1

11

11

0.85

0.87

0.97

0.67

0.69 0.99

0.97

1

1

1

1

1

11

1

1

1

1

1

Brachyelytrum erectumAchnatherum inebriansGlyceria striataElymus canadensis

Hordeum brevisubulatumHordelymus eurupaeusBromus ramosusBromus erectusBromus purgansLolium sp. P4078Lolium sp. P4074Lolium arundinaceumLolium multiflorumLolium edwardiiLolium perenneFestuca rubraFestuca longifolia

Holcus mollisAgrostis teniusAgrostis hiemalisCalamagrostis villosaEchinopogon ovatusKoeleria cristataSphenopholis obtusataBrachypodium sylvaticum

0.01 substitutions/site 0.01 substitutions/site

N. lolli

E. brachyelytriN. gansuenseE. glyceriae

N. sp. HbTG1

E. bromicolaE. bromicola

N. sp. HeTG1

E. elymiE. elymi

N. sp. FaTG2N. sp. FaTG3N. coenophialumN. occultansE. baconii

E. festucaeE. festucaeE. festucaeEpichlo sp. E. amarillansE. amarillansE. baconiiN. aotearoaeN. typhinumE. typhinaE. sylvatica

0.98

0.99

0.97

0.620.63

1

1

1

1

11

1

1

1

1

1

11

11

0.85

0.87

0.97

0.67

0.69 0.99

0.97

1

1

1

1

1

11

1

1

1

1

1

Brachyelytrum erectumAchnatherum inebriansGlyceria striataElymus canadensis

Hordeum brevisubulatumHordelymus eurupaeusBromus ramosusBromus erectusBromus purgansLolium sp. P4078Lolium sp. P4074Lolium arundinaceumLolium multiflorumLolium edwardiiLolium perenneFestuca rubraFestuca longifolia

Holcus mollisAgrostis teniusAgrostis hiemalisCalamagrostis villosaEchinopogon ovatusKoeleria cristataSphenopholis obtusataBrachypodium sylvaticum

0.01 substitutions/site 0.01 substitutions/site

N. lolli

E. brachyelytriN. gansuenseE. glyceriae

N. sp. HbTG1

E. bromicolaE. bromicola

N. sp. HeTG1

E. elymiE. elymi

N. sp. FaTG2N. sp. FaTG3N. coenophialumN. occultansE. baconii

E. festucaeE. festucaeE. festucaeEpichlo sp. E. amarillansE. amarillansE. baconiiN. aotearoaeN. typhinumE. typhinaE. sylvatica

0.98

0.99

0.97

0.620.63

1

1

1

1

11

1

1

1

1

1

11

11

0.85

0.87

0.97

0.67

0.69 0.99

0.97

1

1

1

1

1

11

1

1

1

1

1

Brachyelytrum erectumAchnatherum inebriansGlyceria striataElymus canadensis

Hordeum brevisubulatumHordelymus eurupaeusBromus ramosusBromus erectusBromus purgansLolium sp. P4078Lolium sp. P4074Lolium arundinaceumLolium multiflorumLolium edwardiiLolium perenneFestuca rubraFestuca longifolia

Holcus mollisAgrostis teniusAgrostis hiemalisCalamagrostis villosaEchinopogon ovatusKoeleria cristataSphenopholis obtusataBrachypodium sylvaticum

0.01 substitutions/site 0.01 substitutions/site

N. lolli

E. brachyelytriN. gansuenseE. glyceriae

N. sp. HbTG1

E. bromicolaE. bromicola

N. sp. HeTG1

E. elymiE. elymi

N. sp. FaTG2N. sp. FaTG3N. coenophialumN. occultansE. baconii

E. festucaeE. festucaeE. festucaeEpichlo sp. E. amarillansE. amarillansE. baconiiN. aotearoaeN. typhinumE. typhinaE. sylvatica

0.98

0.99

0.97

0.620.63

1

1

1

1

11

1

1

1

1

1

11

11

0.85

0.87

0.97

0.67

0.69 0.99

0.97

1

1

1

1

1

11

1

1

1

1

1

LolC

NH2

N

CO2H

LolF

N

NH2HO2C

N

NH2

N

H NH2

LolO or LolE

LolT LolD

N

H NH2

OH N

H NH2X

N

H NH2

Oor

norloline

LolO orLolE

NH2

AcO

=

CO2HCO2H

CO2-

NO

NH2

HseOAc

CO2-

L-Proline

+NHCH

H2C

H2CCH2

CO2H

H

H

H

H

HH

HH

HH

Pro

NH2

N*

CO2H

NH2

CO2H

CO2H

HseOAc

O

O

H3C

NH*

CO2HLolC

NH2

HS

NH2

CO2H

CO2H

HseOAc

O

O

H3C

HseOAc (thiol) lyase

H2S

NH2

S

NH2

CO2H

CO2H

HseOAc

O

O

H3C

Cystathionine synthase

NH2SHHO2C

Cys

NH2HO2C

Cystathionine Homocysteine

Methionine Lolines

Ostrecoccus tauriOstrecoccus lucimarinus

Neosartorya fischeri (paralog)

Neosartorya fischeri

Neotyphodium uncinatum LolC1N. uncinatum LolC2

Epichlo festucae LolC

Coccidioides immitisAspergillus clavatusAspergillus fumigatus

Emericella nidulansAspergillus nidulans CysD

Aspergillus nigerAspergillus oryzae

Phaeosphaeria nodorumNeurospora crassa

Gibberella zeae

Chaetomium globosumMagnaporthe grisea

Yarrowia lipolyticaDebaryomyces hansenii

Ustilago maydisCoprinopsis cinerea

domain Bacteria

phylum Basidiomycota

phylum Ascomycota

LolC

NH2

N

CO2H

LolF

N

NH2HO2C

N

NH2

N

H NH2

LolO or LolE

LolT LolD

N

H NH2

OH N

H NH2X

N

H NH2

Oor

norloline

LolO orLolE

NH2

AcO

=

CO2HCO2H

CO2-

NO

NH2

HseOAc

CO2-

L-Proline

+NHCH

H2C

H2CCH2

CO2H

H

H

H

H

HH

HH

HH

1 2 now

MRCAG(1,2)

3 4

A

now

MRCAG(1,2)

1 2 3 4

B

1 2 3' 3 4

D

now

MRCAG(3',4)

MRCAG(3,4)

1 2 3' 3 4

C

now

MRCAG(3',4)

MRCAG(3,4)

Ostrecoccus tauriOstrecoccus lucimarinus

Neosartorya fischeri (paralog)

Neosartorya fischeri

Neotyphodium uncinatum LolC1N. uncinatum LolC2

Epichlo festucae LolC

Coccidioides immitisAspergillus clavatusAspergillus fumigatus

Emericella nidulansAspergillus nidulans CysD

Aspergillus nigerAspergillus oryzae

Phaeosphaeria nodorumNeurospora crassa

Gibberella zeae

Chaetomium globosumMagnaporthe grisea

Yarrowia lipolyticaDebaryomyces hansenii

Ustilago maydisCoprinopsis cinerea

domain Bacteria

phylum Basidiomycota

phylum Ascomycota

NCH3

HN

H

H

CH2OH

HN

C

H

OHN

O

N NO

OH

O

H3C

H

NCH3

HN NH2

CO2H

CO

PPiH

H

HN NH2

CO2H

H H

H

HN NH2

CO2H

H

H

L-Trp

DMAPP

DMATrp

fumigaclavine A

N

HN

HCH3

CH3

HOH3C

O

O

N

HN

HCH3

CH3

HOH3C

O

fumigaclavine C

reverseprenyl-

transferase

PPi

NH

HN

O

OHO

NH

N

NH

O

O

NH

N

HN CO2H

NH2

CO

PPiH

H#

HN CO2H

NH2

H

H#

L-Trp

DMAPP

dmaTrp

*

HNHN

H

chanoclavine-I

CO2HNHCH3 H NHCH3

* OH

NHCH3

HN

C*

H

chanoclavine-I-aldehyde

O H

H

agroclavine

N*

HN

CH3

H

HCH3

elymoclavine

N

HN

H

HCH3

dmaW

CH2OH

ergovaline

N

HN

H CH3

cloACO2H

spont.?N

HN

CH3

C

H

OHN

O

NN

O O

H3C

CH3H3C

OH

H

L-Ala L-Val L-Pro

lpsB + lpsA

D-lysergic acid