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Table 1

A. oryzae strains used in this study.

Strain

Genotype

RIB40

Wild-type

Reference

Higuchi et

NSRku70-1-1A

niaD- sC- adeA- adeA ΔargB Δku70::argB

NSRku70-11AN

al. (2009a)

Higuchi et

niaD–::niaD sC– adeA– adeA ΔargB Δku70::argB

al.(2011)

niaD–::(PaipA-aipA-TaipA niaD) sC– adeA–

Higuchi et

ΔaipA::adeA ΔargB Δku70::argB

al.(2011)

niaD–::(PamyB-aipA niaD) sC– adeA–::adeA

Higuchi et

ΔargB Δku70::argB

al.(2011)

niaD–::(PamyB-aipAK542A niaD) sC– adeA–::adeA

Higuchi et

ΔargB Δku70::argB

al.(2011)

niaD-::(PamyB-aipAE596Q niaD) sC- adeA-::adeA

Higuchi et

ΔargB Δku70::argB

al.(2011)

niaD– niaD sC–::(PamyB-egfp-aipA AosC)

Higuchi et

adeA–::adeA ΔargB Δku70::argB

al.(2011)

DAIPA1-com

PaaA1

PaaA5421

PaaA5961

PaEaASO1

38

niaD-::niaD sC-::(PamyB-egfp-aipAK542A AosC)

Higuchi et

adeA-::adeA ΔargB Δku70::argB

al.(2011)

niaD-::niaD sC-::(PamyB-egfp-aipAE596Q AosC)

Higuchi et

adeA-::adeA ΔargB Δku70::argB

al.(2011)

PaEaA542SO1

PaEaA596SO1

Higuchi et

DAIPA1

niaD- sC- adeA- ΔaipA::adeA ΔargB Δku70::argB

NSRku70-1-

al.(2011)

niaD-::niaD sC-::AosC adeA-::adeA ΔargB

This study

1ANSO

Δku70::argB

niaD-::niaD sC- adeA- ΔaipA::adeA ΔargB

DAIPA1-N

This study

Δku70::argB

niaD-::niaD sC-::AosC adeA- ΔaipA::adeA ΔargB

DAIPA1-NS

This study

Δku70::argB

niaD– ::(PamyB-egfp-aipA niaD) sC–::AosC adeA-

DAIPAS1-PaEA

This study

ΔaipA::adeA ΔargB Δku70::argB

niaD– ::(PamyB-Aoabp1-mdsred niaD) sC–

DABP1-PaAD

This study

ΔAoabp1::AosC adeA–::adeA ΔargB Δku70::argB

39

niaD– ::(PamyB-egfp-aipA niaD) sC–

DAIPAABP1ΔAoabp1::AosC adeA– ΔaipA::adeA ΔargB

This study

PaEA

Δku70::argB

niaD– ::(PamyB-Aoabp1-mdsred niaD) sC–

DAIPAABP1ΔAoabp1::AosC adeA– ΔaipA::adeA ΔargB

This study

PaAD

Δku70::argB

NSRku70-1-1AS niaD– sC–::AosC adeA–::adeA ΔargB Δku70::argB

This study

niaD– sC–::AosC adeA– ΔaipA::adeA ΔargB

DAIPAS1

This study

Δku70::argB

DAIPAS1-

niaD–::(PamyB-egfp-aipAK542A niaD) sC–::AosC

This study

PaEA542

adeA– ΔaipA::adeA ΔargB Δku70::argB

DAIPAS1-

niaD–::(PamyB-egfp-aipAE596Q niaD) sC–::AosC

This study

PaEA596

adeA– ΔaipA::adeA ΔargB Δku70::argB

DAIPAS1-

niaD–::(PamyB-egfp-aipAΔ346-370 niaD)

This study

PaEA346-370

sC–::AosC adeA– ΔaipA::adeA ΔargB Δku70::argB

niaD– sC– ΔAoabp1::AosC adeA– adeA ΔargB

DABP1

This study

Δku70::argB

40

niaD– sC– ΔAoabp1::AosC adeA– ΔaipA::adeA

DAIPAABP1

This study

ΔargB Δku70::argB

niaD–::niaD sC–::AosC adeA–::adeA ΔargB

Can1E

This study

Δku70::argB ptrA::(Aocan1-egfp ptrA)

niaD–::niaD sC–::AosC adeA– ΔaipA::adeA ΔargB

DAIPA1-Can1E

This study

Δku70::argB ptrA::(Aocan1-egfp ptrA)

DAIPA1-N-

niaD-::niaD sC- adeA- ΔaipA::adeA ΔargB

This study

Can1E

Δku70::argB ptrA::(Aocan1-egfp ptrA)

DAIPA1-N-

niaD-::niaD sC-::AosC adeA- ΔaipA::adeA ΔargB

This study

Can1E-p

Δku70::argB ptrA::(Aocan1-egfp ptrA)

niaD-::niaD sC-::(PaipA-aipA-TaipA AosC) adeA-

DAIPA1-NΔaipA::adeA ΔargB Δku70::argB ptrA::(Aocan1-

This study

Can1E-com

egfp ptrA)

niaD-::niaD sC-::(PaipA-aipAK542A-TaipA AosC)

DAIPA1-NadeA- ΔaipA::adeA ΔargB Δku70::argB

Can1E-com542

ptrA::(Aocan1-egfp ptrA)

41

This study

niaD-::niaD sC-::(PaipA-aipAE596Q-TaipA AosC)

DAIPA1-NadeA- ΔaipA::adeA ΔargB Δku70::argB

This study

Can1E-com596

ptrA::(Aocan1-egfp ptrA)

niaD–::(PaipA-aipA-TaipA niaD) sC– adeA–

DAIPA1-comΔaipA::adeA ΔargB Δku70::argB ptrA::(Aocan1-

This study

Can1E

egfp ptrA)

niaD–::(PamyB-aipA niaD) sC– adeA–::adeA

PaaA1-Can1E

This study

ΔargB Δku70::argB ptrA::(Aocan1-egfp ptrA)

PaaA5421-

niaD–::(PamyB-aipAK542A niaD) sC– adeA–::adeA

This study

Can1E

ΔargB Δku70::argB ptrA::(Aocan1-egfp ptrA)

PaaA5961-

niaD-::(PamyB-aipAE596Q niaD) sC- adeA-::adeA

This study

Can1E

ΔargB Δku70::argB ptrA::(Aocan1-egfp ptrA)

NSRku70-1-

niaD–::(Aocan1-egfp niaD) sC–::AosC

This study

1AS-Can1E

adeA–::adeA ΔargB Δku70::argB

niaD–::(Aocan1-egfp niaD) sC– ΔAoabp1::AosC

DABP1-Can1E

This study

adeA–::adeA ΔargB Δku70::argB

DABP1-Can1E-

niaD–::(Aocan1-egfp niaD) sC– ΔAoabp1::AosC

This study

adeA–::adeA ΔargB Δku70::argB ptrA::ptrA

42

niaD–::(Aocan1-egfp niaD) sC– ΔAoabp1::AosC

DABP1-Can1EadeA–::adeA ΔargB Δku70::argB ptrA::(PAoabp1-

This study

com

Aoabp1-TAoabp1 ptrA)

DAIPAABP1-

niaD–::(Aocan1-egfp niaD) sC– ΔAoabp1::AosC

This study

Can1E

adeA– ΔaipA::adeA ΔargB Δku70::argB

NSRku70-1-

niaD–::(PpgkA-Lifeact-egfp niaD) sC–::AosC

This study

1AS-LAE

adeA–::adeA ΔargB Δku70::argB

niaD–::(PagkA-Lifeact-egfp niaD) sC–::AosC

DAIPAS1-LAE

This study

adeA– ΔaipA::adeA ΔargB Δku70::argB

niaD–::(PpgkA-Lifeact-egfp niaD) sC–

DABP1-LAE

This study

ΔAoabp1::AosC adeA– adeA ΔargB Δku70::argB

niaD–::(PpgkA-Lifeact-egfp niaD) sC–

DAIPAABP1ΔAoabp1::AosC adeA– ΔaipA::adeA ΔargB

This study

LAE

Δku70::argB

niaD- ::(PpgkA-egfp-Aosnc1 niaD) sC-::AosC

PpES

This study

adeA-::adeA ΔargB Δku70::argB

niaD-::(PpgkA-egfp-Aosnc1 niaD) sC-::AosC adeA-

DAIPAS1-PpES

This study

ΔaipA::adeA ΔargB Δku70::argB

43

niaD-::(PpgkA-egfp-Aosnc1 niaD) sCDABP1-PpES

This study

ΔAoabp1::AosC adeA-::adeA ΔargB Δku70::argB

niaD-::(PpgkA-egfp-Aosnc1 niaD) sC-

DAIPADABP1ΔAoabp1::AosC adeA- ΔaipA::adeA ΔargB

PpES

Δku70::argB

617

44

This study

618

Table 2 Primers used in this study.

Name

Sequence (5' to 3')

KK134

GATTTAGTTCCGTTCGTGCAGG

KK217

ATGGGGTGACGATGAGCCGC

KK260

GCTATCAACGCGGCCGCTATTGGGCATAAGTATTAGGCGCATTTG

KK261

AACTAAATCGCGGCCTTTCAGAAAGATTCCACAAGGCACAAC

KK262

GTTCCTTGGGCGGCCTCCAAGGTGGTGTCTTCCAC

KK263

TGCACCATAGCGGCCGCATTGATCTTGGCCGCAAGAG

KK305

CCCGGGGTTGATAGCTTGGCGTAATCATG

CAAGCTATCAACCCCGGGATACTTGGGATTTACTTATTGGACTAACCC

KK312

AG

KK313

CTTGCTCACCATCCCGCCTCGAAACGAGCGAAAGATC

KK314

AATCAATTGCCCCCCATCAGACGTTTAAAGTACCCACG

KK315

GAGTGCACCATACCCGGGACCGGAACGGTATAGTCTATGC

KK318

CCAGTAGCAACTTTATTATACATAGTTGATAATTCACTGGC

KK319

GATGCCATCTGTGGGGTTTATTGTTCAGAGAAG

KK320

CCCACAGATGGCATCCCTTAACCTTTCATC

45

KK321

TGTCCATCTTTCGAAGTTCTACATAATTTGCTGG

KK322

TTCGAAAGATGGACAACACCGAGGACGTCATC

KK323

TAAAGTTGCTACTGGGAGCCGGAGTGGC

KK346

ATGATGCGTCCCAAACCGGC

KK347

CTGTGGGGTTTATTGTTCAGAGAAGGGAG

KK348

CAATAAACCCCACAGATGGTGAGCAAGGGCGAGGAG

KK349

TTTGGGACGCATCATCTTGTACAGCTCGTCCATGCCGTG

RH14

TTTCAGGGACCCGGGATGATGCGTCCCAAACCGGC

RH14

TTCTTCTTCGTTGTTCTGTCCTTCGAAGTAGACGGCAGTC

RH14

AACAACGAAGAAGAAGCTTT

RH14

CCGTCTGTAAGTTGGACTAA

RH14

CCAACTTACAGACGGCTTCTCAGGCTCCGACATAA

46

RH14

GGCTTTGTTTAGCAGCTATCCACCTCTCTCGCCGA

RH15

ACGGGTGCCACGATGCTTGCGCGCGCG

RH15

CATCGTGGCACCCGTCCCTGGAGGTCC

RH15

GTGGACCAGATCGACTCACTATTGTCC

RH15

GTCGATCTGGTCCACAAAGATGATCGA

RH16

ATGGGTGTCGCCGATCTCATC

RH16

ATCGGCGACACCCATTGTTCTATCACACAAGGTGGGG

RH16

CTATCAACGCGGCCGCCCATTCTTTATGTTACTTCTATCCTG

47

RH17

GAACGGAACTAAATCCTTGACGGGTTTGTAGTGGAAG

RH17

AAGGATACTGTTCAGTCCGGAGGATCTGCGGGTGC

RH17

CGCAGATCCTCCGGACTGAACAGTATCCTTGGAGCC

RH17

GCTATCAACCCCGGGTATTGGGCATAAGTATTAGGCG

RH17

CTCATCGTCACCCCATATTGATCTTGGCCGCAAGAG

SH21

TATGGTGCACTCTCAGTACAATCTGC

SH22

TATGCGGTGTGAAATACCGCACAG

YHK1

CGTCGAGTCCACTGGTGTCTT

96

YHK1

TTGTTGACACCCATAACGAACATGG

97

48

YHK2

ATCAAGAAGTTCGAGTCCATCTCCAAGGTGGTGATGGTGAGCAAGGG

51

CGAGGAG

YHK2

CTCGAACTTCTTGATGAGATCGGCGACACCCATCTGTGGGGTTTATTG

52

TTCAGAGAAGGG

YHK2

GCTGCAACTACCTCGAACAACC

55

YHK2

TTGTCGCTCAACGCATCTTCAC

56

YM16

ATTTCACACCGCATAGGGGATCTGTAGTAGCTCGTGA

YM17

TGAGAGTGCACCATACGCTTAACAAGTATGATCGTCT

YM56

GATAACAATTTCACATATTGACTACTATGGTAACCAACGCG

YM60

TGTGAAATTGTTATCCGCTGGTATCAG

YM83

GCGGCCGCGTTGATAGCTTGGCG

pET50

b inf

CTGCTAAACAAAGCCCGAAAGGAAGC

Fw

49

pET50

b inf

CCCGGGTCCCTGAAAGAGGACTTCAAG

Rv

619

50

620

Figure legends

621

Fig 1 Localization and endocytosis of AoCan1-EGFP in the ΔaipA strain.

622

(A) The images of representative hyphae of each strain were taken by confocal

623

microscopy at 0, 30, 60, 90 min after the medium shift. Scale bar = 10 µm.

624

(B) The ratio of hyphae with AoCan1-EGFP endocytosed was measured at ten time

625

points for each strain. The error bars represent the SDs from three independent

626

experiments, each with n = 50. *, **Statistically significant difference at P < 0.05, 0.01,

627

respectively (Student’s t test). a, b, c: Statistically significant difference at α = 0.05

628

(tukey-kramer’s multiple test).

629

630

Fig 2 Localization and endocytosis of AoCan1-EGFP in the ΔAoabp1 strain.

631

(A) The images of representative hyphae of each strain were taken by confocal

632

microscopy at 0, 30, 60, 90 min after the medium shift. Scale bar = 10 µm.

633

(B) The ratio of hyphae with AoCan1-EGFP endocytosed was measured at ten time

634

points for each strain. The error bars represent the SDs from three independent

635

experiments, each with n = 50. *, **Statistically significant difference at P < 0.05, 0.01,

636

respectively (Student’s t test). a, b, c: Statistically significant difference at α = 0.05

637

(tukey-kramer’s multiple test).

51

638

639

Fig 3 Localization of Lifeact-EGFP in aipA and Aoabp1 deletion strains.

640

(A) 1.0×105 conidia of each strain in 100 µL of CD medium were cultured at 30°C for 20

641

h, and subsequently observed under fluorescence microscope. Scale bar = 10 µm.

642

(B) Fluorescence intensity profiles of Lifeact-EGFP in each strain were taken at the

643

cross section of 2 µm from the hyphal tip.

644

645

Fig 4 Localization of EGFP-AipA and AoAbp1-mDsRed in ΔaipA and ΔAoabp1

646

cells.

647

(A) The images of representative hyphae of each strain expressing EGFP-AipA or

648

AoAbp1-mDsRed were taken by confocal microscopy. Note that EGFP-AipA was

649

dispersed from the hyphal tip region in the ΔAoabp1 cells. (B) EGFP-AipAΔ346-370

650

exhibited cytoplasmic localization. (C) EGFP-AipAE542A showed similar localization to

651

EGFP-AipA; in contrast, EGFP-AipAE596Q exhibited both apical and basal localization.

652

Scale bar = 10 µm.

653

654

Fig 5 Localization and endocytosis of AoCan1-EGFP in the ΔaipAΔAoabp1 strain.

655

(A) The images of representative hyphae of each strain were taken by fluorescent

52

656

microscopy at 0, 30, 60, 90 min after the medium shift. Scale bar = 10 µm.

657

(B) The ratio of hyphae with AoCan1-EGFP endocytosed was measured at ten time

658

points for each strain. The error bars represent the SDs from three independent

659

experiments, each with n = 50. *Statistically significant difference at P < 0.05 (Student’s

660

t test).

661

662

Fig 6 Localization and endocytosis of AoCan1-EGFP in aipA-, aipAK542A- and

663

aipAE596Q-overexpressing strains.

664

(A) The images of representative hyphae of each strain were taken by confocal

665

microscopy at 0, 30, 60, 90 min after the medium shift. Scale bar = 10 µm.

666

(B) The ratio of hyphae with AoCan1-EGFP endocytosed was measured at ten time

667

points for each strain. The error bars represent the SDs from three independent

668

experiments, each with n = 50. a, b, c: Statistically significant difference at α = 0.05

669

(tukey-kramer’s multiple test).

670

671

Fig 7 Localization of EGFP-AipA in the aipA-overexpressing background.

672

Hyphal images of each strain were taken by confocal microscopy at approximately 30

673

min after the treatment of NOC or its solvent DMSO. Scale bar = 10 µm.

53

674

675

Fig 8 Localization and endocytosis of AoCan1-EGFP in aipA-, aipAK542A-and

676

aipAE596Q-complementary strains.

677

(A) The images of representative hyphae of each strain were taken by confocal

678

microscopy at 0, 30, 60, 90 min after the medium shift. Scale bar = 10 µm.

679

(B) The ratio of hyphae with AoCan1-EGFP endocytosed was measured at ten time

680

points for each strain. The error bars represent the SDs from three independent

681

experiments, each with n = 50. a, b: Statistically significant difference at α = 0.05

682

(tukey-kramer’s multiple test).

683

684

Fig 9 Schematic diagram of AoCan1 endocytosis in A. oryzae.

685

The rate of AoCan1 endocytosis is depicted as the width of the arrow (red, accelerated;

686

blue, delayed) in WT and each mutant strain.

54

Fig. 1 Hiasa et al.

(A)

0 min

30 min

60 min

90 min

0 min

30 min

60 min

90 min

Control

ΔaipA

(B) 120

Cells with endocytosis (%)

Control

Control

100

ΔaipA

DaipA

80

60

40

20

20

40

60

Time (min)

80

100

Fig. 2 Hiasa et al.

(A)

0 min

30 min

60 min

0 min

90 min

comp

Aoabp1

Control

0 min

30 min

60 min

90 min

ΔAoabp1

(B)

120

Cells with endocytosis (%)

Control

100

ΔAoabp1

DAoabp1

compAoabp1

80

60

ab

ab

ab

ab

40

ab

20

20

40

60

Time (min)

80

100

30 min

60 min

90 min

Fig. 3 Hiasa et al.

Lifeact-EGFP

(A)

Control

ΔaipA

ΔAoabp1

ΔaipAΔAoabp1

(B) Control

ΔaipA

Intensity

Intensity

Length

ΔAoabp1

Length

ΔaipAΔAoabp1

Intensity

Intensity

Length

18

Length

Fig. 4 Hiasa et al.

Control

(A)

Control

DIC

DIC

EGFP-AipA

AoAbp1mDsRed

ΔAoabp1

ΔaipA

DIC

DIC

EGFP-AipA

AoAbp1mDsRed

(B)

DIC

EGFPAipAΔ346-370

(C)

DIC

DIC

EGFPAipAK542A

EGFPAipAE596Q

Fig. 5 Hiasa et al.

(A)

0 min

30 min

60 min

90 min

0 min

30 min

60 min

90 min

Control

ΔaipA

ΔAoabp1

(B)

120

Cells with endocytosis (%)

Control

100

ΔaipAΔAoabp1

DaipADAoabp1

80

60

40

20

20

40

60

Time (min)

80

100

Fig. 6 Hiasa et al.

0 min

(A)

30 min

60 min

90 min

Control

30 min

60 min

90 min

aipAK542A

O/E

aipAE596Q

O/E

(B)

Cells with endocytosis (%)

120

100

80

60

40

ab

ab

ab

ab

ab

ab

ab

ab

cont.

Control

aipA

O/E

aipA

O/E

aipAK542A

aipAK542A O/E

ab

ab

30 min

60 min

90 min

0 min

30 min

60 min

90 min

aipA O/E

0 min

20

0 min

bc

20

E596Q O/E

aipA

aipA

E506Q

O/E

40

60

Time (min)

80

100

Fig. 7 Hiasa et al.

DMSO

DIC

EGFPAipA

DIC

EGFPAipAK542A

DIC

EGFPAipAE596Q

Nocodazole

Fig. 8 Hiasa et al.

(A)

0 min

30 min

60 min

90 min

30 min

60 min

90 min

0 min

30 min

60 min

90 min

comp

aipA

Control

(ΔaipA)

0 min

30 min

60 min

90 min

comp

aipAK542A

comp

aipAE596Q

(B)

100

Cells with endocytosis (%)

0 min

80

60

control(ΔaipA)

cont.

(ΔaipA)

40

compaipA

compaipA

compaipAK542A

compaipAK542A

20

compaipAE596Q

compaipAE596Q

20

40

60

Time (min)

80

100

Fig. 9 Hiasa et al.

(A) WT

AipA

AipA

AipA

AipAK542A

endocytic vesicle

AipA

AipAE596Q

endocytosis

AoAbp1

AoAbp1

AoAbp1

AipA

(B) ΔaipA

(E) aipA O/E

(H) ΔaipA + aipA

AoAbp1

AoAbp1

AoAbp1

AipA

AipA

AipA

AipA

(F) aipAK542A O/E

(C) ΔAoabp1

(I) ΔaipA + aipAK542A

AoAbp1

AoAbp1

AipA

AipA

AipA

AipA

AipA

(D) ΔaipAΔAoabp1

(G) aipAE596Q O/E

(J) ΔaipA + aipAE596Q

AoAbp1

AipA

AoAbp1

AipA

AipA

AipA

Supplementary materials

Involvement of AAA ATPase AipA in endocytosis of the arginine permease

AoCan1 depending on AoAbp1 in Aspergillus oryzae

Reiko Hiasa, Ken-ichi Kakimoto, Kaoru Takegawa, Yujiro Higuchi*

Department of Bioscience and Biotechnology, Faculty of Agriculture, Kyushu University,

744 Motooka, Fukuoka 819-0395, Japan

10

*Corresponding author. Tel/Fax: +81 92 802 4734, E-mail address:

11

y.higuchi@agr.kyushu-u.ac.jp

12

13

Word count: Abstract, 250; Main, 5519.

14

Number of figures: Main, 9; Supplementary, 5.

15

Number of tables: Main, 2.

16

Supplementary figure legends

17

Fig S1 Localization of EGFP-AoSnc1 in deletion strains.

18

Approximately 1.0×105 conidia of each strain in 100 µL of CD medium were cultured at

19

30°C for 20 h, and subsequently observed under confocal microscope. Scale bar = 10

20

µm.

21

22

Fig S2 Growth test of deletion strains.

23

Approximately 1.0×104 conidia of each strain were inoculated onto PD or M medium

24

plate added with the indicated reagent, and were cultured at 30, 37°C for 4 days.

25

26

Fig S3 AAA ATPase assay of AipA.

27

AAA ATPase activity was measured using the recombinant AipA. The vertical axis is the

28

relative ratio with the ATPase activity of WT=1. The error bars represent the SDs from

29

three independent experiments, each with n=3.

30

31

Fig S4 Expression analysis of aipA-, aipAK542A- and aipAE596Q-overexpressing

32

strains.

33

Relative expression levels of aipA in cells of each strain cultured with maltose,

34

normalized by the expression of gpdA. The relative ratios are depicted with the

35

expression level of the control strain as 1. The error bars represent the SDs from three

36

independent experiments, each with n=3.

37

38

Fig S5 Expression analysis of aipA-, aipAK542A- and aipAE596Q-complementary

39

strains.

40

Relative expression levels of aipA in cells of each strain cultured in CD medium,

41

normalized by the expression of gpdA. The relative ratios are depicted with the

42

expression level of the aipA-complementary strain as 1. The error bars represent the

43

SDs from three independent experiments, each with n=3.

44

45

46

47

48

...