The fungal vision

Light regulation in fungi and strategies to study them

Reinhard Fischer
Institute for Applied Biosciences, Dept. of Microbiology

KIT University of the State of Baden-Wrttemberg and

National Large-scale Research Center of the Helmholtz Association

www.kit.edu

Vitamin B2 (Riboflavin)
Die kleinsten Mitarbeiter der BASF

x 1.000.000
x300

Vitamin B2 - Ashbya gossypii

Wildtyp Ashbya gossypii

Mutante

Vitamin B2

Aspergillus niger

Zitronensure

Waschen bei 30C - Pilz machts mglich!

Pilze wachsen auf Polymeren!

Neurospora crassa!

Nick Read, Edinburgh

Pilze als Lebensmittel - oder Gift!

Brain Development in Human!

Lissenzephalie!

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

McConnell 1995, Neuron 15, 761!

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!
Pathogene!

Destruenten!

Symbiosen!

Lebensmittel-!
technologie!
!
Biotechnologie!

Importance of fungi!

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

Light
O2

Wind

Temperature shifts
Osmotic stress
High ROS

Temperature shifts buffered

Humidity high

Low O2

Darkness
Low ROS
Nick Read, Edinburgh

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

Circadian clocks in fungi

Circadian clocks in fungi

Phototropism in Phycomyces

Luis Corrochano, Sevilla, Spain

Idnurm et al., 2006, PNAS, 103:4546-4551

Light sensing
LOV-Photoreceptors
Phototropins
Neochromes
ZTL
White-Collar1

Cryptochrome

400

Plants
Animals
Fungi

500

Plants
Animals
Fungi
Bacteria

Phytochrome

Opsin

600

700 nm

Plants
Animals
Fungi
Bacteria

Fungi
Bacteria

19

Oidiation is induced by blue light

Action spectrum

2x108

white control

dark control

710-900 nm

600-760 nm

510-770 nm

480-600 nm

360-490 nm

1x108

300-370 nm

oidia per culture

3x108

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

Neurospora crassa

Blue-light responses in
Neurospora crassa
Bl ueLi ght
light adaptation
vvd
(initial)
FAD

?
WC-1
PWC-2
P

kinases
incl. PKC

phototropism
?
circadian rhythm
frq
photoconidiation
con-6, con-10
perithecial beak bending
?

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)

Isolation of blind mutants

Forward genetics

mycelial carot. biosynthesis

al-1, al -2, al-3

Jay C. Dunlap
G. Macino

Blue-light regulation in
Neurospora crassa
Bl ueLi ght
light adaptation
vvd
(initial)
FAD

?
WC-1
PWC-2
P

kinases
incl. PKC

phototropism
?
circadian rhythm
frq
photoconidiation
con-6, con-10
perithecial beak bending
?

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)

mycelial carot. biosynthesis

al-1, al -2, al-3

Jay C. Dunlap
G. Macino

H. Linden, Science, 2002

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

Development in Aspergillus nidulans

asexual

sexual

Action spectrum for light-mediated !

conidiation!
Log light intensity

300

400

500
Wavelength (nm)

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

600

700

Pr,
Inactive
r

Pr!

Pfr, active

dr!

Pfr!
Strasburger, 2002

Action spectrum for light-mediated !

conidiation!
Log light intensity

300

400

500
Wavelength (nm)

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

600

700

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

Plant

S/T

Cyanobacteria

Bacteria

Fungi

PAS2

HKRD
ATP

ATP

ATP

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 IX

Nicole Frankenberg-Dinkel

0.14

absorbance!

Pr!
0.12

0.10

Pfr!

absorbance

0.08

0.06
660

680

700

720

740

wavelength [nm]

760

780

800

Phytochrome structures
Photosensory domain

Signal output domain

P1 P2/PAS P3/GAF P4/PHY PAS1

Plant

S/T

Cyanobacteria

Bacteria

PAS2

Fungi

HKRD
ATP

ATP

ATP

ATP

RR

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

Fungal phytochromes are more !

similar to bacterial phytochromes!
189
218
234
14
5
18
93
9
10
11
9
9
63
63

V
G
G
T
T
E
E
Q
D
N
T
S

D
E
P
P
I
F
V
V
Q
I
S
D

S
T
V
K
K
T
D
L
D
S
N
S
K

F
L
L
L
L
T
P
L
L
L
L
V
A
V

K
Q
Q
D
D
E
S
A
A
S
R
T
V
T

A
R
R
S
S
N
I
N
N
N
Q
S
S
T

C
C
C
C
C
C
C
C
C
C
L
L
A
T

E
E
E
G
G
E
E
E
A
S
E
K
Y
Y

D
D
D
A
A
R
M
D
D
K
T
E
I
L

E
E
E
E
E
E
E
E
E
E
L
A
Q
H

P
P
P
P
P
P
P
P
P
P
A
P
N
H

I
I
I
I
I
I
I
I
I
I
I
I
M
I

H
H
H
H
H
H
A
H
Q
H
H
H
Q
Q

I
L
T
I
I
I
T
V
F
I
T
L
R
K

P
P
P
P
P
P
P
P
P
P
A
S
G
G

G
G
G
G
G
G
G
G
G
G
H
G
R
K

A
A
A
A
A
S
A
A
A
L
L
Q
Y
L

I
V
I
I
I
I
I
I
I
I
I
I
I
I

Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q
Q

S
G
T
E
E
P
P
P
P
P
P
P
P
P

F
F
F
H
H
H
H
H
H
H
H
H
F
F

G
G
G
G
G
G
G
G
G
G
G
G
G
G

A
L
A
A
A
A
A
A
L
V
L
V
C
C

L
L
L
L
L
L
L
L
L
L
V
L
L
L

V
V
V
L
L
L
M
V
F
L
V
L
L
L

A
A
A
V
V
T
T
T
T
V
V
V
A
A

V
L
L
L
L
A
A
L
L
L
L
L
I
L

R
R
R
S
S
D
R
R
K
Q
Q
E
H
D

499
529
543
245
236
255
330
242
251
255
255
255
319
319

A
A
A
S
S
A
S
S
S
S
S
S
A
A

M
M
M
V
V
T
V
V
V
V
A
A
P
P

S
S
S
S
S
S
S
S
S
S
Y
A
H
H

P
P
P
P
P
P
P
P
P
P
H
N
G
S

I
I
I
V
V
M
V
I
I
L
C
C
C
C

H
H
H
H
H
H
H
H
H
H
H
H
H
H

I
L
I
L
L
M
L
C
C
I
L
L
A
L

K
K
K
E
E
Q
D
E
Q
E
T
E
Q
Q

Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y
Y

L
L
L
M
M
L
M
L
M
M
L
L
M
M

A
A
K
R
R
R
Q
T
K
Q
K
H
A
A

N
N
N
N
N
N
N
N
N
N
N
N
N
N

M
M
M
M
M
M
M
M
M
M
M
M
M
M

Q
A
A
G
G
G
N
G
G
G
G
G
G
D

I
V
V
T
T
V
T
V
V
V
V
V
S
S

R
R
R
A
A
G
A
R
L
T
G
G
I
I

0.02

FphA!

0.01

0.00

absorbance

-0.01

FphAC195A!

-0.02
500

550

600

650

700

wavelength [nm]

750

800

S
S
S
A
A
S
A
A
S
A
A
A
A
A

S
S
S
S
S
S
S
S
S
S
S
S
S
S

M
M
M
M
M
L
L
M
M
M
L
L
L
L

S
S
S
S
S
S
T
S
S
S
T
T
V
V

I
I
I
V
V
V
I
I
V
I
I
I
M
M

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

FphA is a red-light sensor in !

A. nidulans!
Reverse genetics !
fphA

dark

wt

fphA

wt

Red light

Proteins involved in light perception

Red and blue control development !

Light inhibits germination in A. nidulans

Julian Rhrig, Christian Kastner

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

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!

Interaction of photoreceptors!
Y

VeA

FphA

VeA phA

FphA and VeA physically

interact IN THE NUCLEUS

GFP-LreB

FphA-HA

Co-IP strain

GFP-LreA

HA

FphA-HA

FphA

Co-IP strain

A light regulator complex in !

A. nidulans!

200

GFP LreB

GFP

VeA
119

GFP

LreA

GFP -LreA

GFP-LreB

66

antibody

43

Anti-GFP

Gene regulation
%&&#'(#

$)&#'(#

*($%
!"#$%
&'()%

&'($%

Light regulated gene

$!#

Spotted microarray light vs. dark (A. nidulans)

Slides from Craig Venter Institute (PFGRC)

!"2 colour assay (Light vs. Dark)

!"3 biological replicates
!"Flip dye control

A. nidulans photoreceptor complex

11 W/m2

White fluorescent lamps

Light regulated genes

Upregulated genes (Top 15)
Top
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
Top
20
1
21
2
22
3
23
4
24
5
25
6
26
7
27
8
28
9
29
10
30
11
31
12
32
13
33
14
34
15
35
16
36
17
37
18
38
19
39
20
40
21
41
22
42

Locus
AN5056
AN9285
AN0045
AN0693
AN8339
AN4299
AN7558
AN8641
AN8638
AN8018
AN9310
AN5004
AN5015
AN3872
AN5764
AN5338
AN6005
AN1547
AN5090
Locus
AN9168
AN1052
AN4483
AN8647
AN2792
AN1008
AN0400
AN5558
AN5217
AN3304
AN2477
AN0231
AN11477
AN8063
AN7590
AN9076
AN1754
AN2926
AN4946
AN8539
AN4911
AN5353
AN8643
AN9240
AN3679
AN0367
AN6881
AN0190
AN10847
AN1131
AN10581
AN10489
AN1331
AN8683
AN0600
AN6503
AN3361
AN8727
AN2901
AN8956
AN1918
AN4648
AN12426*
AN2388
AN10422

GeneN
grg1 (circadian rhythm early morning expressed transcript) homologue (ccgB )
grg1 (circadian rhythm early morning expressed transcript) homologue (ccgA )
solid-state culture expressed protein (Aos23), putative
Hypothetical protein
Hypothetical protein
Clock-controlled and temperature-regulated gene
Hypothetical protein
Hypothetical protein
Conidia Enriched Transcript (cetJ)
auxin efflux transporter family protein
Hypothetical protein
Hypothetical protein
conidiation gene (conJ )
Hypothetical protein
Hypothetical protein
flavin-binding monooxygenase-like protein
Hypothetical protein
Putative acetyl-CoA hydrolase with a predicted role in acetate, acetaldehyde, and ethanol (two-carbon) metabolism (acuH)
Hypothetical
protein
GeneN
conserved
hypothetical
protein - putative
sugar
Protein involved
in light-sensitive
control
oftransporter
differentiation and secondary metabolism (VeA)
calcium/calmodulin-dependent protein kinase, putative
ALS family protein;ALS family includes cell-surface glycoproteins, some with adhesin function
Hypothetical protein
Putative nitrate transporter with 10 predicted tran segments; regulated by NirA, AreA,and NiaD (crnA)
Hypothetical protein
alkaline protease (prtA)
primary component of eisosomes
GABA transporter, putative
plasma membrane channel protein (Aqy1), putative
Conidiophore-specific phenol oxidase (ivoB)
Hypothetical protein
acid phosphatase activity
Putative reductase with a predicted role in carbohydrate metabolism/L-xylulose reductase
putative adhesin function
Hypothetical protein
60S ribosomal protein Nsa2, putative
conserved serine-rich protein
GNAT family acetyltransferase, putative
Hypothetical protein
Hypothetical protein
bZIP transcription factor (Atf21), putative
Putative C2H2 finger domain transcription factor
oxidoreductase, short-chain dehydrogenase/reductase family
integral membrane protein
Hypothetical protein
Subunit of the tRNA splicing endonuclease,
Hypothetical protein
cytosolic Cu/Zn superoxide dismutase, putative
integral membrane protein
FKBP-type peptidyl-prolyl cis-trans isomerase
Hypothetical protein
Putative ferric reductase with similarity to Fre2p; expression induced by low copper levels
Hypothetical protein
Putativebacteriorhodopsin
transcription factor
with
zinc
putative
/opsin
(nop
A)finger DNA-binding motif C2H2
Hypothetical
protein
Putative arginase with a predicted role in arginine metabolism (aga 1)
phosphate-repressible
phosphate
permease,
Putative
phosphoenolpyruvate
carboxykinase
with aputative
predicted role in gluconeogenesis and glycolysis (acu F)
small
nucleolar
ribonucleoprotein
complex
subunit Utp15, putative
stress response RCI peptide
endoglucanase,
Hypothetical
proteinputative

Downregulated genes (Top 15)

log2 ratio dark vs light

-7,90
-7,78
-7,75
-7,26
-6,81
-6,55
-6,36
-6,27
-6,19
-6,07
-6,07
-6,02
-5,95
-5,93
-5,75
-5,72
-5,61
-5,47
-5,47
log2 ratio
dark vs light
-5,44
3,11
-5,34
2,65
-5,23
2,37
-5,16
2,31
-5,14
2,20
-5,03
2,14
-4,95
1,98
-4,92
1,97
-4,84
1,95
-4,75
1,92
-4,72
1,89
-4,70
1,81
-4,69
1,80
-4,58
1,77
-4,57
1,75
-4,50
1,75
-4,50
1,72
-4,50
1,68
-4,49
1,63
-4,47
1,62
-4,45
1,60
-4,44
1,56
-4,36

Fold change
239,35
219,79
214,97
152,75
112,13
93,57
82,03
77,33
73,06
67,37
67,32
65,03
61,82
60,84
53,67
52,56
48,84
44,45
44,42
Fold
change
43,41
8,60
40,45
6,28
37,48
5,17
35,78
4,95
35,14
4,59
32,60
4,40
30,97
3,94
30,17
3,91
28,68
3,86
26,93
3,78
26,35
3,70
25,99
3,49
25,83
3,49
23,98
3,41
23,77
3,36
22,69
3,36
22,66
3,30
22,61
3,21
22,53
3,09
22,12
3,08
21,81
3,04
21,77
2,96
20,48

Light regulated genes

Total genes
(last annotation August 2009)

10560
260

Light controlled genes

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

51
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

Up and Down

2.46%

19.61%

Total genes

97.53%

Light controled
genes

Downregulated
>2,23 fold
80.39%

Upregulated
>3,49 fold

Light
Temperature
shifts

O2

Wind

Osmotic stress
High ROS

Temperature
shifts buffered
Humidity high
Darkness
Low ROS

Low O2

Gene regulation
%&&#'(#

$)&#'(#

*($%
!"#$%
&'()%

&'($%

Light regulated genes: ccgA, conJ

$*#

Isolation of novel components

(Zhenzhong Yu)
Relative expression level

ccgA
350
300
250
200
150
100
50
0

!"#$%
!"#$
*($%

ccgA
&'()%

dark

light

!"#$%
!"#$
*($%
*($

pyrG

&'()%

&'($%

ccgA

&'($%

No growth

pyrG

Growth

Mutagenesis
UV

!"#$%
!"#$
*($%

!"#$%
!"#$
*($%
*($
&'()%

&'($%

ccgA

ccgA

pyrG
&'()%

Growth

pyrG

&'($%

No growth
Blind mutant

Isolation of blind mutants

Dark

Wildtype

U/U

without
U/U

parent

Light

Wildtype

parent

Dark/without U/U

Isolation of blind mutants

Isolation of novel components

Dark

parent

Light

M1-22

M8-17

Without
U/U

:(;<=>(%(?"'(@@ABC%;(>(;%

!"#$%
)&&#
$&&#
,&&#

-./0#

+&&#

12345#

!&&#
&#

/0*-+1% /23-4% 5-6..% 5768% 596-4%

.+++%
-,++%
F<'G%

-+++%

&AE#H%

,++%
+%

/0*-+1% /23-4% 5-6..% 5768% 596-4%

DBCIJ"KLL"M'1%
:(;<=>(%(?"'(@@ABC%;(>(;%

M6-9

:(;<=>(%(?"'(@@ABC%;(>(;%

DDE$%

With
U/U

-.++%
-+++%
9++%

F<'G%

7++%

&AE#H%

1++%
.++%
+%

/0*-+1% /23-4% 5-6..%

5768%

596-4%

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

Summary!
H

ATP

RR

Phytochrome in fungi!
Light regulator complex!
Development and secondary !
metabolism share regulators!

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-Wrttemberg priority programme

Food and Health, Humboldt Society, Centre for functional nanostructures (CFN)