A Comparative Genomic Investigation of Fungal Genome Evolutionstajichlab.fungalgenomes.org/presentations/stajich_10Apr2006... · A Comparative Genomic Investigation of Fungal Genome

A Comparative Genomic Investigation

of Fungal Genome Evolution

Jason StajichDuke University

University Program in Genetics & Genomics

Comparative Genomics

Evolutionary genomics

Evolution & Organismal

PhenotypePopulation structureEcological adaptationNiche changesPhylogeny

Model Systems

Genetic toolsGene function &

expressionRegulatory networksPathwaysMolecular & cellular

biologyDisease models

Molecular evolutionGene orderGene familiesGene and genome

structureGene contentConserved elementsRates of molecular

evolution

Power of the comparative approach

A B A B C D E

??

Industrial uses of fungi

• Bread, beer, wine - Saccharomyces cerevisiae

• Sake and soy sauce - Aspergillus oryzae

• Dairy - Penicillium roqueforti, Kluyveromyces lactis

• Citric acid - Aspergillus niger

• Riboflavin - Ashbya gossypii

• Stonewashed jeans - Trichoderma reesei

• Penicillin antibiotic - Penicillium notatum

Agricultural impact of fungi

• Two-thirds of plant disease is caused by fungi

• Wheat blight (Fusarium)

• Strawberry grey mold (Botrytis)

• Leaf rusts (Puccinia)

• Wheat and maize smuts (Ustilago).

• Also deposit mycotoxins - e.g. ergot

• Mycorrhizal fungi provide nutrient exchange and nitrogen fixation

www.gov.mb.ca

USDA A.G. Bölker

Impact of fungi on human health

• Biggest risk for immunocompromised individuals

• Primary pathogens

• Histoplasma, Coccidioides, Cryptococcus gattii

• Opportunistic pathogens

• Candida albicans, Aspergillus fumigatus, Cryptococcus neoformans, Rhizopus oryzae

Fungi as genetic models

• Beadle and Tatum (1941) - one gene, one enzyme hypothesis in Neurospora crassa

• Cell cycle, cell model - Saccharomyces cerevisiae and Schizosaccharomyces pombe

• Straightforward molecular biology tools to investigate phenotype-genotype

• Evolutionary models

Fungal genomes

• Smaller than most vertebrate and plant genomes

• A. gossypii 8.5 Mb, S. cerevisiae 12 Mb

• N. crassa 40 Mb

• Animals: 100 Mb worm, 3000 Mb Human

• Vary in protein coding gene content

• 4700 in A. gossypii, 5800 in S. cerevisiae

• 16,000 in R. oryzae or S. nodorum

• 19,000 in Fruitfly, 25,000 in worm

Fungal comparative genomics

A B C D E

Recent Intron Lossin C.neoformans

Evolution of fungal introns

Animals Fungi

Fungal gene family evolution

Central dogma of eukaryotic biology

Introns

splicing to form mRNA

Genomic DNA

Ribosome

Protein

mRNA

mRNA

ExonExonExonIn

tro

n

pre-mRNA

Genomic DNA

Protein

Intr

on

Orthologs and Paralogs

Speciation

Gene

Duplication

Tim

e

Paralogs

V W

V

U

Orthologs

A

A A

A1 A2

Genome annotation

• Many of the fungal genomes were only assembled genomic sequence.

• Automated annotation pipeline was built to generate to get systematic gene prediction.

• Several gene prediction programs were trained and results were combined to produce composite gene calls

SNAPTwinscan

GlimmerGenscan

ZFF to GFF3

GFF2 to GFF3

Tools::Genscan

Tools::Glimmer

BLASTZBLASTN

SearchIO

Bio:

:DB:

:GFF

HMMER

GFF to AA

Genome

Proteins

predicted proteins

SearchIO

ProteinsFASTAall-vs-all

exonerateprotein2genome

GFF2 to GFF3

Findorthologs

protein to genome

coordinates

GLEAN(combiner)

Tools::GFF

exonerateest2genome

ESTs

SearchIO

MCL

Gene families

Rfam tRNAscan

Multiple sequence alignment

Intron mapping into

alignmentIntron analysis

Bio::AlignIOaa2cds alignment

AnalysisMethodology

http://fungal.genome.duke.edu


A B C D E

Recent intron lossin C. neoformans


Animals Fungi


Evolution of gene structure

• Present day introns

• Recent insertions?

• Introns late hypothesis

• Formed in eukaryotic ancestor?

• Introns early hypothesis / exon theory of genes

• Mixture of two?

Previous work on intron evolution

• Rogozin et al. 2003

• 7 genomes

• 684 genes, 7236 positions

• Other methods

• Roy and Gilbert. 2005

• Csũrös. 2005

• Nguyen et al. 2006

Calculating intron densities across a phylogeny

Basidiomycota

Archiascomycota

Hemiascomycota

Euascomycota

Opisthokont

Vertebrates

Plants

Zygomycota

Chaetomium globosum (463)Neurospora crassa (336)

Podospora anserina (359)Magnaporthe grisea (368)

Fusarium graminearum (372)Aspergillus fumigatus (481)Aspergillus terreus (474)Aspergillus nidulans (469)

Stagonospora nodorum (403)Saccharomyces cerevisiae (7)Candida glabrata (6)

Kluyveromyces lactis (6)Ashbya gossypii (7)

Debaryomyces hansenii (5)Yarrowia lipolytica (30)

Schizosaccharomyces pombe (214)Phanerochaete chrysosporium (1615)Coprinus cinereus (1621)

Cryptococcus neoformans (1578)Ustilago maydis (86)

Rhizopus oryzae (947)Mus musculus (2658)Homo sapiens (2737)Fugu rubripes (2687)

Arabidopsis thaliana (2290)

0.1 substitutions

Intron frequency varies among the fungi

1 2 3 4 5 60

100

200

300

400

500

Mean Introns per gene

Me

an

In

tro

n le

ng

th

Rory

Umay

SpomCneo

PchrCcin

Cgla

Klac

Ylip

Scer

Dhan

Agos

Pans

Analysis of whole genomes

• 25 entire genomes

• 21 fungi, 3 vertebrates, 1 plant

• Largest dataset ever assembled for intron analysis

• 1160 orthologous genes

• 7533 intron positions

• 4.15 Mb coding sequence (CDS) per genome

7533 intron positions

25 s

peci

es

predicted proteins

Proteins

FASTAall-vs-all

Findorthologs

Multiple sequence alignment

Intron mapping into

alignmentIntron analysis

Analysis Methods

ATG GTG CGT ACA

Intron phase

ATG GT G CGT ACA

ATG G TG CGT ACA

phase 0

phase 1

phase 2

exon exonintron

Conserved intron positions

Patterns of conservation

Introns late Introns early(ier)

7533 Intron positions 7533 Intron positions

25 s

peci

es

animalsplant

Fung

i

Intron positions shared with animals or plants

S.

ce

revis

iae

Y.

lipo

lytica

F.

gra

min

ea

rum

M.

grise

a

P.

an

se

rin

a

N.

cra

ssa

C.

glo

bo

su

m

A.

nid

ula

ns

A.

terr

eu

s

A.

fum

iga

tus

S.

no

do

rum

R.

ory

za

e

U.

ma

yd

is

C.

ne

ofo

rma

ns

C.

cin

ere

us

P.

ch

ryso

sp

oriu

m

S.

po

mb

e

C.

gla

bra

ta

A.

go

ssyp

ii

K.

lactis

D.

ha

nse

nii

Percentage

45

40

35

30

25

20

15

10

5

0

Phylogenetic signal in intron positions

afum

ater

anid

snod

cglo

ncra

pans

mgri

fgra

agos

klac

cgla

scer

dhan

ylip

spom

ccin

pchr

cneo

umay

rory

frub

hsap

mmus

atha

afum

ater

anid

cglo

pans

ncra

fgra

mgri

snod

agos

cgla

klac

dhan

scer

ylip

umay

spom

ccin

pchr

cneo

rory

frub

hsap

mmus

atha

Strict consensus tree of16 parsimonious trees using

dollo parsimony based on 7533 intron positionsMaximum Likelihood tree based on alignment of 30 proteins

Species Tree30 proteins

Parsimony reconstruc

tion

Intron position reconstruction

• 3 Methods

• Roy and Gilbert. 2005

• Csũrös. 2005

• Nguyen et al. 2006

• Methods agree for all but 2 nodes in tree

Reconstruction of ancestral intron densities

A.

tha

lian

a

R.

ory

za

e

U.

ma

yd

is

C.

ne

ofo

rma

ns

C.

cin

ere

us

P.

ch

ryso

sp

oriu

m

S.

po

mb

e

So

rda

rio

myce

tes

Eu

rotio

myce

tes

+

S.

no

dsu

m

Y.

lipo

lytica

Sa

cch

aro

myce

s

Ve

rte

bra

tes

5.51 6.61 2.28 0.21 3.80 3.89 3.90 0.52 0.89 1.12 0.07 0.02

1.20

3.55

3.55

4.01 0.07

1.50

2.20

3.50

3.72

4.674.67 Introns

per kb of CDS

Exon length of 214 bp

Conclusions

• Early eukaryotic crown genes were complex!

• Ancestor had 70% of the introns in vertebrates

• More introns than previously reported

• Intron loss has dominated among the fungi

• Hemiascomycota experienced loss

• Sampling can bias interpretations - all fungi are not equal.


A B C D E



Animals Fungi


Mechanism of intron loss

• S. cerevisiae and Hemiascomycota have undergone intron loss.

• How are introns lost from the genome?

• Are they lost independently?

• Are they lost many at a time?

• Molecular mechanism of loss

Models of intron loss

• All introns in S. cerevisiae are in 5’ end of gene

• G. Fink proposed transcripts recombine with genome 3’ -> 5’ explaining 5’ retention bias.

• In S. cerevisiae most intron loss events occurred too long ago so little evidence supporting any mechanism

Sequenced Cryptococcus genomes

0.1

C. gattii, strain WM276

C. gattii, strain R265

C. neoformans var. neoformans, strain JEC21

C. neoformans var. grubii, strain H99

substitutions

Screen for intron changes

• Annotate 3 Cryptococcus genomes (var. grubii and 2 var. gattii genomes)

• Identify and align 4-way orthologous genes

• 5298 orthologous genes (out of ~6500)

• Identify intron position changes

Stajich and Dietrich. Euk Cell In press.

Intron loss in var. grubii

0.1

1kb 2 kb 3 kb 4 kb 5 kb 6 kb

JEC21

WM276

R265

H99

2462

35-23

BT-100

BT-63

BT-157C. gattii, strain WM276




A

B

C

1 2 3 4 5 6 7 8 9-19

1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 18 19 20 21 22

20 21 22

0.1

1kb 2 kb 3 kb 4 kb 5 kb 6 kb

JEC21

WM276

R265

H99

2462

35-23

BT-100

BT-63

BT-157C. gattii, strain WM276




A

B

C

1 2 3 4 5 6 7 8 9-19

1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 18 19 20 21 22

20 21 22


CNI01550 - putative RNA helicase

var. grubii

neof

orm

ans

gatti

i

gatti

i

Intron loss was a precise excision

Intron Missing


Other examples of loss

PRP8 homolog (Splicing)

1,3 beta-glucan synthase (cell wall)

Ubiquitin protein ligase (protein degradation)


CNN02320/FKS1A

5 61 2 3 4 7 8 9 10 11 12

3-7 8 9 10 11 121 2

JEC21

R265

WM276

H99

CNG04610

3 4

5.1 5.2 6

C

JEC21

R265

WM276

H99

3 4

65

CNA01350

5 6

1 2 3 4-6 7 8 9 10 11 12 13 14 15

B

JEC21

R265

WM276

H991 2 3 4 7 8 9 10 11 12 13 14 15

LOSS

LOSS

GAIN

Conclusions

• Intron loss via homologous recombination with spliced transcript

• Large losses are all adjacent introns

• Precise deletion

• Loss biased towards the middle of gene not 3’


A B C D E



Animals Fungi


Gene family evolution

• Gene families are the crucible of new genes and thus new functions

• Signature of adaptive evolution often confounded in multi-gene families

• Can we identify families that are have unexpectedly large changes in size across a phylogeny?

• Follow up these families with more focused studies

Identifying family expansions

• Previous work only considered pairwise

• Ad hoc comparison of gene family sizes

• C.elegans-C.briggsae - GPCR family expansions (Stein et al, PLOS Biology 2004)

• A. gambiae-D. melanogaster - Mosquito specific family expansions related to symbiotic bacteria (Holt et el, Science 2002).

Gene family sizes follow power law distribution

N.crassaA.gossypiiR.oryzaeA.oryzaeA.terreusC.cinereusU.maydis

101

1

10

100

1000

10000

100

Family size

Fre

quency o

f F

am

ily s

ize

Multicopy genesSugar transporters

P450 Enzymes

single copy genesPRP8 (splicing)

CDC48 (cell cycle ATPase)

Fully sequenced fungal genomesRhizopus oryazeNeurospora crassaPodospora anserinaChaetomium globosumMagnaporthe griseaFusarium verticillioidesFusarium graminearumTrichoderma reeseiSclerotinia sclerotiorumBotrytis cinereaStagonospora nodorumUncinocarpus reesiiCoccidioides immitisHistoplasma capsulatumAspergillus fumigatusAspergillus nidulansAspergillus terreusAspergillus oryzaeAshbya gosspyiiKluyveromyces lactisSaccharomyces cerevisiaeCandida glabrataCandida lusitaniaeDebaryomyces hanseniiCandida guilliermondiiCandida tropicalisCandida albicansCandida dubliniensisYarrowia lipolyticaSchizosaccharomyces pombeCryptococcus neoformansCryptococcus neoformans H99Cryptococcus gattii WM276Cryptococcus gattii R265Phanerochaete chrysosporiumCoprinus cinereusUstilago maydis

Saprophyte

Bread mold, Opp Hum pathogen

Saprophyte

Hemibiotroph - Rice

Hemibiotroph - wheat

Primary Hum pathogen

Primary Hum pathogen

Opp Hum pathogen

Saprophyte/Industrial uses

Hemibiotroph - maize

Saprophyte

Necrotroph

Necrotroph - fruits

Hemibiotroph - wheat

Opp Hum pathogen

Biotroph/Industrial uses

Industrial uses

Industrial uses

Opp Hum pathogen

Opp Hum pathogen

Opp Hum pathogen

Opp Hum pathogen

Opp Hum pathogen

Opp Hum pathogenSaprophyte

SaprophyteBiotroph - maize

Opp Hum pathogen

Opp Hum pathogen

Opp HumOpp Hum pathogen

Industrial uses

SaprophyteOpp Hum Pathogen

Saprophyte

pathogen

0100200300400500600700800900Million

years ago

Euascomycota

Zygomycota

Hemiascomycota

Archiascomycota

Basidiomycota

Phylogenetic evaluation of gene family size change• Previous methods only used ad hoc

statistics

• Explicit model for gene family size change according to a Birth-Death models

• Apply BD to family size along phylogeny using probabilistic graph models

• CAFE - Computational Analysis of gene Family Evolution Hahn et al, Genome Res 2005

De Bie, et al Bioinformatics 2006Demuth et al, submitted

A B C D E

FASTAall-vs-all

MCL

Gene families

CAFE

18 U. maydis

5 C. gattii R265

5 C. gattii WM276

5 gattii

5 C. neoformans JEC21

5 C. neoformans var grubii

5 neoformans

5 Cryptococcus

163 P. chrysosporium

141 C.cinereus

136 Homobasidiomycota

23 Hymenomycota

23 Basidiomycota

0100200300400

Family 1 P < 0.001 Branch A

Family 2 P < 0.001 Branch B

Family 3 P=0.02 Branch C,E

Family 4 P=0.03 Branch D

10 1 2

14 18 2

7 1 1

6 1 12

6 1 8

3 1 1+

Family count

Spec

ies

Families with significant expansionsVitamin & Cofactor transport

Lactose & sugar transport

Amine transport

Myo-instol, quinate, and glucose transport

Oligopeptide transport

ABC transporter

MFS, drug pump, & sugar transport

Transport

Monocarboxylate & sugar transport

ABC transport

Amino acid permease

Methytransferase

Cytochrome P450: CYP64

Cytochrome P450: CYP53,57A

Cytochrome P450

Kinase

Subtilase family

NADH flavin oxidoreductase

Aldehyde dehydrogenase

Aldo/kedo reductase

Multicopper oxidase

AMP-binding enzyme

TransportersKinasesP450

Oxidation

Transporters

• Of 45 significant families, 22 were related to transport

• Vitamin and amino acid transport

• Sugar and sugar-like transporters

• Multidrug and efflux pumps

• ABC transporters (ATP Binding Cassette)

Branches with transporter expansions

• Sugar related, Drug pump, and MFS

• Aspergillus spp, Fusarium spp, S. nodorum

• Euascomycota

• Vitamin transport

• C.neoformans, Fusarium

• A. nidulans (Biotin)

Aspergillus

Fusarium

S. nodorum

C. neoformans

What do phytopathogens use transporters for?

• Sugar transporters are used to extract nutrients from host

• Haustorium: specialized structure for plant parasitism

• Many sugar transporters highly and specifically expressed in haustoria

Haustorium

http://tolweb.org/Robert Bauer

FIG

.1.

The

exte

rnal

and

inte

rnal

stru

ctur

esof

C.n

eofo

rman

sar

esh

own

bym

eans

ofa

mod

ified

Indi

ain

kpr

epar

atio

n.M

agni

ficat

ion,

ca.!

1,00

0.

Con

tinue

dfro

mpr

eced

ing

page

2291

Cryptococcus sugar transporters

• 3x as many sugar transporters in C. neoformans than other basidiomycetes

• “sugar coated killer”

• Capsule is mixture of glucose, xylose, and mannose.

Zerpa et al, 1996

P450 CYP64

18 U. maydis

5 C. gattii R265

5 C. gattii WM276

5 gattii

5 C. neoformans JEC21

5 C. neoformans var grubii

5 neoformans

5 Cryptococcus

163 P. chrysosporium

141 C.cinereus

136 Homobasidiomycota

23 Hymenomycota

23 Basidiomycota

0100200300400

P450 enzymes involved in synthesis and cleavage of chemical bonds. Drug metabolism in animals.

CYP64: Step in Aspergillus spp aflatoxin pathwayP. chrysosporium implicated in lignin and hydrocarbon degradation.

Million yearsago

CYP64 was from independent duplication

ccin

03995 ccin

12432

ccin

12477

ccin

12447

ccin

03760 ccin

08843

ccin

08880

ccin

08948

ccin

08949

ccin

09228

ccin

08947

ccin

08946

ccin

12431

ccin

12515

ccin

08608

ccin

12514

ccin

07535

ccin

07536

ccin

05141

ccin

07531

ccin

04462

ccin

04461

ccin

04460

ccin

07538

ccin

01326

ccin

04884

ccin

07555

ccin

07554

ccin

03994

ccin

12516

ccin

09337

ccin

09357

ccin

09950

ccin

00042

ccin

00039

ccin

00043

ccin

11079

ccin

11073

ccin

12301

ccin

03618

ccin

12868

ccin

12386

ccin

03622

ccin

09244

ccin

08520

ccin

10950

ccin

13218

ccin

13220

pchr

04215

pchr

02481

pchr

02475

pchr

02324

pchr

02322

pchr

02461

pchr

02249

pchr

02248

pchr

02471

pchr

02460

pchr

02469

pchr

02472

pchr

02470

pchr

02468

pchr

02462

pchr

02442

pchr

02441

pchr

02317

pchr

02473

pchr

02477

pchr

02480

pchr

02479

pchr

02474

pchr

02459

pchr

02478

pchr

09197

pchr

08602

pchr

02326

pchr

08048

pchr

08046

pchr

08045

pchr

08047

pchr

02328

pchr

06733

tree 1

6195

pchr

10861

pchr

07430

pchr

07443

C. cinereus expansionP. chrysosporium

expansion

Tom VolkMario Cervini

Local duplications created CYP64 expansion

9k 10k 11k 12k 13k 14k 15k 16k 17k 18k 19k 20k 21k 22k 23k 24k

pchr_24pchr_24

GLEAN modelsGLEAN_02414

Probability 1

GLEAN_02415

Probability 0.999937

GLEAN_02416


GLEAN_02417


Pfam domainsp450

Cytochrome P450 evalue:1e-28

p450 p450


p450p450

Cytochrome P450 evalue:6.3e-23

p450p450


p450

http://fungal.genome.duke.edu

Family size contractions

• Histoplasma, Coccidioides many families

• Hemiascomycetes - P450

• C. neoformans - P450

• U. maydis - Lactose transport

Conclusions

• Sugar transporters are highly expanded in independent lineages

• Saprophytic and phytopathogenic lifestyles

• P450 CYP64 independent expansions in Homobasidiomycetes

• Lignin degradation and saprophytic lifestyles

• Family size contractions among lineages containing primary pathogens

• Genome streamlining?

Overall conclusions

• Multiple genome sequences have helped resolve several outstanding questions in evolution introns

• Gene family expansions can be important in identifying molecular basis for adaptation

Future directions

• UC Berkeley with John Taylor

• Adaptation and speciation in fungi

• Focus on pathogenic fungus Coccidioides

• Signatures of adaptation among genomes of 12 sequenced strains

AcknowledgmentsFred DietrichGreg Wray

Lincoln SteinJohn McCuskerAlex Hartemink

Joseph HeitmanMarcy Uyenoyama

Rytas VilglyasTim JamesPat Pukkilla

Robert CramerJames FraserSteven GilesAlex IdnurmScott Roy

Dave Des MaraisHeath O’BrienMatt Rockman

MDGDietrich LabAndria Allen

Stephanie DiezmannCharles HallShan HuangPhilippe Lüdi

Laura KavanaughSandra ReynoldsMark DeLong

UPGG

Ewan Birney

DUMRUJohn Perfect

Andy AlspaughTom Mitchell

Sequencing centersBroad InstituteDuke University

Joint Genome InstituteGénolevures

Stanford University(NIH and NSF)

Matthew Hahn

Documents

A Comparative Genomic Investigation of Fungal Genome Evolutionstajichlab.fungalgenomes.org/presentations/stajich_10Apr2006... · A Comparative Genomic Investigation of Fungal Genome