KIT – University of the State of Baden-Württemberg and National Large-scale Research Center of the Helmholtz Association

Institute for Applied Biosciences, Dept. of Microbiology

www.kit.edu

The fungal vision

Light regulation in fungi and strategies to study them

Reinhard Fischer

Die kleinsten Mitarbeiter der BASF

x300

x 1.000.000

Vitamin B2 (Riboflavin)

Vitamin B2 - Ashbya gossypii

Wildtyp Ashbya gossypii Mutante

Vitamin B2

Aspergillus niger

Zitronensäure

Waschen bei 30°C - Pilz machts möglich!

Pilze wachsen auf Polymeren!

Nick Read, Edinburgh Neurospora crassa!

Pilze als Lebensmittel - oder Gift!

Brain Development in Human!

McConnell 1995, Neuron 15, 761!

Lissenzephalie!!- smooth cortex!- neuronal migration defect!!- mentally retarded!- short life expectation!!!

Lis-1 displays homology to A. nidulans nudF!

Cell Translocation Requires Nuclear Migration

Klominek 1991, PNAS 88, 3902

Nuclear migration in a fungus

Importance of fungi!

Lebensmittel-!technologie!

Symbiosen!

Pathogene!

! Biotechnologie!

Destruenten!

Importance of fungi!

http://www.zdf.de/ZDFmediathek/beitrag/video/1133648/Pilze

Light

Darkness Low ROS

Humidity high

Temperature shifts buffered

Low O2

Wind

Temperature shifts

O2

Osmotic stress High ROS

Rodriguez et al., 2010, Ann. Rev. Microbiol. 65:585-610 Nick Read, Edinburgh

Circadian clocks in fungi

Circadian clocks in fungi

Luis Corrochano, Sevilla, Spain Idnurm et al., 2006, PNAS, 103:4546-4551

Phototropism in Phycomyces

400 500 600 700 nm

Phytochrome

LOV-Photoreceptors Phototropins Neochromes

ZTL White-Collar1

Cryptochrome Opsin

Light sensing

Animals Fungi

Bacteria

Plants Animals

Fungi

Plants Plants Animals

Fungi Bacteria

19  

Fungi Bacteria

300-

370

nm

360-

490

nm

480-

600

nm

510-

770

nm

600-

760

nm

710-

900

nm

dark

con

trol

whi

te c

ontr

ol

oidi

a pe

r cul

ture

1x10 8

2x10 8

3x10 8

Oidiation is induced by blue light Action spectrum

Kertesz-Chalupkova, K. (1998) Fungal Genet. Biol., 23:95-109

Neurospora crassa

?

B l u e L i g h t

P P W C - 1

P P P

W C - 2

a l - 1 , a l - 2 , a l - 3 m y c e l i a l c a r o t . b i o s y n thesis

c o n - 6 , c o n - 1 0

f r q c i r c a d i a n r h y t h m

p h o t o c o n i d i a t i o n

v v d l i g h t a d a p t a t i o n

F A D

? p e r i t h e c i a l b e a k b e n d i n g

? p h o t o t r o p i s m

? (initial)

kinases incl. PKC

Summary of light responses in Neurospora. (from Dunlap, J. C. and Loros, J. J. 2005. Neurospora photoreceptors, Handbook of Photosensory Receptors, ed. W. R. Briggs and J. Spudich. Wiley-VCH, in press. Figure courtesy of C. Schwerdtfeger)

Jay C. Dunlap G. Macino

Blue-light responses in Neurospora crassa

Isolation of blind mutants Forward genetics

?

B l u e L i g h t

P P W C - 1

P P P

W C - 2

a l - 1 , a l - 2 , a l - 3 m y c e l i a l c a r o t . b i o s y n thesis

c o n - 6 , c o n - 1 0

f r q c i r c a d i a n r h y t h m

p h o t o c o n i d i a t i o n

v v d l i g h t a d a p t a t i o n

F A D

? p e r i t h e c i a l b e a k b e n d i n g

? p h o t o t r o p i s m

? (initial)

kinases incl. PKC

Summary of light responses in Neurospora. (from Dunlap, J. C. and Loros, J. J. 2005. Neurospora photoreceptors, Handbook of Photosensory Receptors, ed. W. R. Briggs and J. Spudich. Wiley-VCH, in press. Figure courtesy of C. Schwerdtfeger)

Jay C. Dunlap G. Macino

H. Linden, Science, 2002 He et al., Froehlich et al., Science 2002

Blue-light regulation in Neurospora crassa

asexual

sexual

Development in Aspergillus nidulans

Mooney and Yager (1990), Genes & Dev. 4:1473-1482

Action spectrum for light-mediated !conidiation!

Wavelength (nm) 300 400 500 600 700

Log

light

inte

nsity

1

0

2

3

Strasburger, 2002

Pr! Pfr!

r dr!

Pr, Inactive

Pfr, active

Mooney and Yager (1990), Genes & Dev. 4:1473-1482

Action spectrum for light-mediated !conidiation!

Wavelength (nm) 300 400 500 600 700

Log

light

inte

nsity

1

0

2

3

Velvet A is a regulator but no phytochrome

Genome Analysis Phytochrome structures

Reverse Genetics approach Photosensory domain Signal output domain

P1 P2/PAS P3/GAF P4/PHY PAS1 PAS2 HKRD

Plant C S/T ATP

Cyanobacteria C ATP H

Bacteria C ATP H C

Fungi ATP H ATP RR

Galagan et al. (2005), Nature, 438:1105-1115 Blumenstein et al. (2005), Curr. Biol., 15:1833-1838

FphA in E. coli!Co-expression with cyanobacterial heme oxygenase!

Heme - biliverdin IXα Nicole Frankenberg-Dinkel!

wavelength [nm]

660 680 700 720 740 760 780 800

ab

so

rban

ce

0.06

0.08

0.10

0.12

0.14

Pr!

Pfr!abso

rban

ce!

Phytochrome structures

Photosensory domain Signal output domain

P1 P2/PAS P3/GAF P4/PHY PAS1 PAS2 HKRD

Plant C S/T ATP

Cyanobacteria C ATP H

Bacteria C ATP H C

Fungi ATP H ATP RR

Galagan et al. (2005), Nature, 438:1105-1115 Blumenstein et al. (2005), Curr. Biol., 15:1833-1838

?

A M S P I H I K Y L A N M Q I R S S M S I 499 A M S P I H L K Y L A N M A V R S S M S I 529 A M S P I H I K Y L K N M A V R S S M S I 543 S V S P V H L E Y M R N M G T A A S M S V 245 S V S P V H L E Y M R N M G T A A S M S V 236 A T S P M H M Q Y L R N M G V G S S L S V 255 S V S P V H L D Y M Q N M N T A A S L T I 330 S V S P I H C E Y L T N M G V R A S M S I 242 S V S P I H C Q Y M K N M G V L S S M S V 251 S V S P L H I E Y M Q N M G V T A S M S I 255 S A Y H C H L T Y L K N M G V G A S L T I 255 S A A N C H L E Y L H N M G V G A S L T I 255 A P H G C H A Q Y M A N M G S I A S L V M 319 A P H S C H L Q Y M A N M D S I A S L V M 319

A. nidulans C. heterotrophus G. moniliformis A. tumefaciens 1 A. tumefaciens 2 D. radiodurans R. spaeroides P. aeruginosa P. syringae Calothrix 7601 B Synechocystis 6803 Calothrix 7601 A T. aestivum A. thaliana A

V D S F K A C E D E P I H I P G A I Q S F G A L V A V R 189 G E T L Q R C E D E P I H L P G A V Q G F G L L V A L R 218 G - V L Q R C E D E P I H T P G A I Q T F G A L V A L R 234 T P K L D S C G A E P I H I P G A I Q E H G A L L V L S 14 T P K L D S C G A E P I H I P G A I Q E H G A L L V L S 5 E I T T E N C E R E P I H I P G S I Q P H G A L L T A D 18 - F D P S I C E M E P I A T P G A I Q P H G A L M T A R 93 - - - L A N C E D E P I H V P G A I Q P H G A L V T L R 9 E V L L A N C A D E P I Q F P G A I Q P H G L L F T L K 10 Q V D L S N C S K E P I H I P G L I Q P H G V L L V L Q 11 D Q S L R Q L E T L A I H T A H L I Q P H G L V V V L Q 9 N I N V T S L K E A P I H L S G Q I Q P H G V L L V L E 9 T S S A V S A Y I Q N M Q R G R Y I Q P F G C L L A I H 63 S D K V T T T Y L H H I Q K G K L I Q P F G C L L A L D 63

*

*

*

Fungal phytochromes are more !similar to bacterial phytochromes!

wavelength [nm]

500 550 600 650 700 750 800

Δ

absorbance -0.02

-0.01

0.00

0.01

0.02

FphA!

FphAC195A!

FphA is a red-light sensor in !A.   nidulans!

Reverse genetics !

∆fphA ∆fphA wt wt

dark Red light

Proteins involved in light perception

Red and blue control development !

Röhrig et al. (2013), Curr. Genet., 59:55-62

Light inhibits germination in A. nidulans Julian Röhrig, Christian Kastner

Blue light represses mycotoxin !production!

FphA localizes to the cytoplasm!Prediction: nuclear!

FphA-GFP! DsRed-Nuclei! Overlay!

LreA, LreB and VeA are !nuclear proteins!

LreA-GFP!

FphA and VeA physically interact IN THE NUCLEUS

phA VeA VeA FphA

Y

Interaction of photoreceptors!

FphA HA

LreB

LreA

VeA GFP

GFP

GFP 200

119

66

43

Co-I

P st

rain

FphA

-HA

GFP

-Lre

B

GFP-LreB

antibody

Anti-GFP

A light regulator complex in !A. nidulans!

Co-I

P st

rain

FphA

-HA

GFP

-Lre

A

GFP -LreA

Gene regulation

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Light regulated gene

Spotted microarray light vs. dark (A. nidulans)

!"2 colour assay (Light vs. Dark) !"3 biological replicates !"Flip dye control

11 W/m2

A. nidulans photoreceptor complex

White fluorescent lamps

Slides from Craig Venter Institute (PFGRC)

Upregulated genes (Top 15) Top Locus GeneN log2 ratio dark vs light Fold change1 AN5056 grg1 (circadian rhythm early morning expressed transcript) homologue (ccgB ) -7,90 239,352 AN9285 grg1 (circadian rhythm early morning expressed transcript) homologue (ccgA ) -7,78 219,793 AN0045 solid-state culture expressed protein (Aos23), putative -7,75 214,974 AN0693 Hypothetical protein -7,26 152,755 AN8339 Hypothetical protein -6,81 112,136 AN4299 Clock-controlled and temperature-regulated gene -6,55 93,577 AN7558 Hypothetical protein -6,36 82,038 AN8641 Hypothetical protein -6,27 77,339 AN8638 Conidia Enriched Transcript (cetJ) -6,19 73,0610 AN8018 auxin efflux transporter family protein -6,07 67,3711 AN9310 Hypothetical protein -6,07 67,3212 AN5004 Hypothetical protein -6,02 65,0313 AN5015 conidiation gene (conJ ) -5,95 61,8214 AN3872 Hypothetical protein -5,93 60,8415 AN5764 Hypothetical protein -5,75 53,6716 AN5338 flavin-binding monooxygenase-like protein -5,72 52,5617 AN6005 Hypothetical protein -5,61 48,8418 AN1547 Putative acetyl-CoA hydrolase with a predicted role in acetate, acetaldehyde, and ethanol (two-carbon) metabolism (acuH) -5,47 44,4519 AN5090 Hypothetical protein -5,47 44,4220 AN9168 conserved hypothetical protein - putative sugar transporter -5,44 43,4121 AN4483 calcium/calmodulin-dependent protein kinase, putative -5,34 40,4522 AN2792 Hypothetical protein -5,23 37,4823 AN0400 Hypothetical protein -5,16 35,7824 AN5217 primary component of eisosomes -5,14 35,1425 AN2477 plasma membrane channel protein (Aqy1), putative -5,03 32,6026 AN11477 Hypothetical protein -4,95 30,9727 AN7590 Putative reductase with a predicted role in carbohydrate metabolism/L-xylulose reductase -4,92 30,1728 AN1754 Hypothetical protein -4,84 28,6829 AN4946 conserved serine-rich protein -4,75 26,9330 AN4911 Hypothetical protein -4,72 26,3531 AN8643 bZIP transcription factor (Atf21), putative -4,70 25,9932 AN3679 oxidoreductase, short-chain dehydrogenase/reductase family -4,69 25,8333 AN6881 Hypothetical protein -4,58 23,9834 AN10847 Hypothetical protein -4,57 23,7735 AN10581 integral membrane protein -4,50 22,6936 AN1331 Hypothetical protein -4,50 22,6637 AN0600 Hypothetical protein -4,50 22,6138 AN3361 putative bacteriorhodopsin /opsin (nopA) -4,49 22,5339 AN2901 Putative arginase with a predicted role in arginine metabolism (aga 1) -4,47 22,1240 AN1918 Putative phosphoenolpyruvate carboxykinase with a predicted role in gluconeogenesis and glycolysis (acu F) -4,45 21,8141 AN12426* stress response RCI peptide -4,44 21,7742 AN10422 Hypothetical protein -4,36 20,4843 AN1278 Hypothetical protein -4,31 19,7844 AN5207 Hypothetical protein -4,30 19,7545 AN3512 Hypothetical protein -4,30 19,7446 AN4711 Hypothetical protein -4,28 19,4147 AN5332 AraC-like ligand binding domain protein, putative -4,23 18,7748 AN8195 Hypothetical protein -4,22 18,6049 AN8591 calcium binding protein Caleosin, putative -4,22 18,5750 AN10135 Hypothetical protein -4,20 18,40

Top Locus GeneN log2 ratio dark vs light Fold change1 AN1052 Protein involved in light-sensitive control of differentiation and secondary metabolism (VeA) 3,11 8,602 AN8647 ALS family protein;ALS family includes cell-surface glycoproteins, some with adhesin function 2,65 6,283 AN1008 Putative nitrate transporter with 10 predicted tran segments; regulated by NirA, AreA,and NiaD (crnA) 2,37 5,174 AN5558 alkaline protease (prtA) 2,31 4,955 AN3304 GABA transporter, putative 2,20 4,596 AN0231 Conidiophore-specific phenol oxidase (ivoB) 2,14 4,407 AN8063 acid phosphatase activity 1,98 3,948 AN9076 putative adhesin function 1,97 3,919 AN2926 60S ribosomal protein Nsa2, putative 1,95 3,8610 AN8539 GNAT family acetyltransferase, putative 1,92 3,7811 AN5353 Hypothetical protein 1,89 3,7012 AN9240 Putative C2H2 finger domain transcription factor 1,81 3,4913 AN0367 integral membrane protein 1,80 3,4914 AN0190 Subunit of the tRNA splicing endonuclease, 1,77 3,4115 AN1131 cytosolic Cu/Zn superoxide dismutase, putative 1,75 3,3616 AN10489 FKBP-type peptidyl-prolyl cis-trans isomerase 1,75 3,3617 AN8683 Putative ferric reductase with similarity to Fre2p; expression induced by low copper levels 1,72 3,3018 AN6503 Putative transcription factor with zinc finger DNA-binding motif C2H2 1,68 3,2119 AN8727 Hypothetical protein 1,63 3,0920 AN8956 phosphate-repressible phosphate permease, putative 1,62 3,0821 AN4648 small nucleolar ribonucleoprotein complex subunit Utp15, putative 1,60 3,0422 AN2388 endoglucanase, putative 1,56 2,9623 AN1500 C2H2 finger domain protein, putative 1,56 2,9524 AN7357 Hypothetical protein 1,56 2,9525 AN11046 Hypothetical protein 1,53 2,8926 AN6221 GATA factor AREB (areB) 1,52 2,8727 AN6703 sugar transporter family protein 1,49 2,8128 AN0418 urea transporter (Dur3), putative 1,49 2,8029 AN3037 carboxylesterase, putative 1,46 2,7530 AN8445 Vacuolar aminopeptidase Y 1,42 2,6731 AN7794 Sensor-transducer of the stress-activated PKC1-MPK1 signaling pathway 1,41 2,6532 AN4770 Putative adenosine 3-phosphate 5-phosphosulfate (PAPS) reductase with a predicted role in cysteine metabolism (sA) 1,38 2,6133 AN0275 lysosomal L-cystine transporter 1,37 2,5834 AN10087* proliferating cell nuclear antigen 1,36 2,5735 AN6413 integral membrane protein 1,35 2,5436 AN8890 cellobiose dehydrogenase, putative 1,34 2,5437 AN10698 putative adhesin function 1,34 2,5438 AN2044 Hypothetical protein 1,32 2,4939 AN2572 hypothetical dipeptidyl-peptidase 1,27 2,4040 AN1145 Hypothetical protein 1,26 2,4041 AN8186 Hypothetical protein 1,26 2,4042 AN1711 nonsense-mediated mRNA decay protein 3 Protein involved in nuclear export of the large ribosomal subunit 1,26 2,3943 AN9407 Putative multifunctional enzyme with a predicted role in cytosolic fatty acid formation (fasA) 1,25 2,3744 AN4260 GPI-anchored protein 1,24 2,3645 AN0822 kfsA/mitochondrial inheritance component mdm12 1,23 2,3546 AN1719 Hypothetical protein 1,23 2,3547 AN5664 Hypothetical protein 1,22 2,3448 AN6973 Putative C-4 sterol methyl oxidase with a predicted role in sterol metabolism 1,22 2,3249 AN6612 ribosomal large subunit biogenesis protein MAK16, putative 1,21 2,3150 AN7914 Putative alcohol dehydrogenase; member of the F9775 secondary metabolite gene cluster 1,20 2,30

Downregulated genes (Top 15)

Light regulated genes

Total genes (last annotation August 2009) 10560 Light controlled genes 260

Down-regulated >2.23 fold 51 Up-regulated >3,49 fold 209

Similar to Neurospora: 3% (Lewis et al. 2002) Spotted array 6% (Chen-Hui Chen,2009) Affimetrix array Similar to Trichoderma: 2.8% (Rosales-Saavedra,2006) Spotted array

Around 3.2% if we set the thresholds in >2,3 fold

Light regulated genes

97.53%

2.46%

Total genes

Light controled genes

19.61%

80.39%

Downregulated >2,23 fold Upregulated >3,49 fold

Up and Down

Light

Darkness Low ROS

Humidity high

Temperature shifts buffered

Low O2

Wind Temperature shifts

O2

Osmotic stress High ROS

Gene regulation

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Light regulated genes: ccgA, conJ

Isolation of novel components (Zhenzhong Yu)

0

50

100

150

200

250

300

350

Rel

ativ

e ex

pres

sion

leve

l

ccgA

dark light light

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pyrG ccgA

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No growth

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Isolation of „blind“ mutants

LightDark

U/U

without U/U

Wildtype parentDark/without U/U

Wildtype parent

Isolation of „blind“ mutants

Isolation of novel components

With U/U

LightDarkparent M1-22

M6-9 M8-17

+%

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16.000 survivors, 336 small colonies tested, 3 with desired phenotype

Phytochrome in fungi!Light regulator complex!

Development and secondary !metabolism share regulators!

Summary!

ATP H ATP RR

Strategies:!•" Action spectra!•" Mutant analysis (forward genetics)!•" Reverse genetics analysis!•" Gene expression analyses!•" Promoter analysis !!

The Fischer lab in Karlsruhe

Sponsors: DFG, Max-Planck, Landesstiftung Baden-Württemberg priority programme Food and Health, Humboldt Society, Centre for functional nanostructures (CFN)