Supporting information pp g - Semantic Scholar

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C. miyabeanus. 55. 100. 0.05 g ... posadasii, C. heterostrophus, Cochliobolus heterostrophus; C. graminicola, Colletotrichum graminicola; C. gloeosporioides ...
Supporting pp g information Albarouki et al. 2014

Table S1. Primers used in this study. Sequence overlaps with the hph are underlined; restriction sites are given in bold. Primer

sequence (5´→3´)

ACT1-qRT-Fw

TCCTACGAGCTTCCTGACGG

ACT1-qRT-Rw

CCGCTCTCAAGACCAAGGAC

CHSIII-qRT-Fw

CACGCCTAGTGACCAACTAC

CHSIII-qRT-Rv

CCTCCGGGTTGACACCATAG

CHSI-qRT-Fw

ATGGCCACAATATGCAGGACC

CHSI-qRT-Rv

CCGAGATGGCTCTCGTAAGG

CHSV-qRT-Fw

CGGAGACGTGCTGAAGATGGC

CHSV-qRT-Rv

AAGGGCTGGATGGCTTCTAAC

EGFP-Fw

GGAGTCCAGTGTCGAAGAGAAC

EGFP-Rv.1

GCTGGTGACGGAATTTTCATAG

EGFP-Rv.2

GGAGTCCAGTGTCGAAGAGAAC

GLS1-qRT-Fw

ACCCAACAGACCGATTCCTAC

GLS1-qRT-Rv

CTGGTGATAGGCACTGTTTGG

H3-qRT.Fw

ATCCGTCGCTACCAGAAGTC

H3-qRT.Rv

TGAAGTCCTGGGCAATCTCAC

Hyg-Fw

ATTCCCCAGGGATACCGAGCTCCCAAATCTGTC

Hyg-prob-Fw

TCCGAGGGCAAAGGAATAGAGTAG

Hyg-Rv

AATAGAAGAGCGGGCGCTTACACAGTACACGAG

NPS2-qRT-Fw

CGCCATTCGATTCTTGGAGTG

NPS2-qRT-Rv

GGTAGTAGCGGAATCGAGAAC

NPS6-Fw

CAGGCATTGACACGAACCATAC

NPS6-KO-nest-Fw

GCCACCCAATCACTCCACTCTC

NPS6-KO-nest-Rv

GCCTCGGAGACGAGCTTCATAG

NPS6-qRT-Fw

ACGCGCAAACAGTACCGGTG

NPS6-qRT-Rv

CTCGACCTGATATGTCCAGTC

NPS6-Rv

ATTCGTTGACGGTGAGCGTGAG

pEB-uni-Fw

GGAAACAGCTATGACCATGATTAC

pEB-uni-Rv

TACGACTCACTATAGGGCGAATTG

SID1-DraIII-SfiIB-Rv

TCGAGCACCATGTGGCCGAGGCGGCCGACATTATGATGATAGTGGTG (DraIII and SfiI)

SID1-FW.1

CGTATAGTGTCTAGCCGCAGTAGC

SID1-KO-3´-Fw

CTCGTGTACTGTGTAAGCGCCCGCTCTTCTATTCGACGCCGGTGTTTATCTGC (hph)

SID1-KO-3´-Rv

GAGCTCATGACGTGGATGGACAAC

SID1-KO-5´-Fw

CGTATAGTGTCTAGCCGCAGTAGC

SID1-KO-5´-Rv

GACAGATTTGGGAGCTCGGTATCCCTGGGGAATGTGACTCGCAGTGAGGAGAC (hph)

SID1-KO-nest-Fw.1

TAGTGTCTAGCCGCAGTAGC

SID1-KO-nest-Fw.2

CTTACCCATCTGCGTCGTTC

SID1-KO-nest-Rv.1

CAGCGACATGCAACTGTATAG

SID1-KO-nest-Rv.2

CTTCAAGCTGCAAGCACAAG

SID1-Kpn2I-SfiIA-Fw

TCGAGTCCGGAGGCCATTACGGCCCGTATAGTGTCTAGCCGCAGTAGC (Kpn2I and SfiI)

SID1-qRT-Fw

ATCGACATGCTCCGCGACAG

SID1-qRT-Rv

GTCGCGGGAAAGATGGAGTC

SID1-SfiIB-Rv

TGTGGCCGAGGCGGCCGTTGGAAACGGCCTTGGCCC (SfiI)

SIT1-SfiIA-Fw

TTAGGCCATTACGGCCCGACGATCCCTCAAGGAATAAAG (SfiI)

SIT1-SfiIB-Rv

TTAGGCCGAGGCGGCCAACAACATTTCCGCGCACCTG (SfiI)

ZmPR1-qRT-Fw

CAACAGCTGGACCCTCGAGATC

ZmPR1-qRT-Rv

AACTGCCTGACGCTGCCAAC

ZmChit-qRT-Fw

ACCGCCTTATTCTTCGCTGTGC

ZmChit-qRT-Rv

AAGCCCGCGTAGGTGTAGAAG

ZmPRX346-qRT-Fw

TTCCTGATGCCACCAAGGGTTC

ZmPRX346-qRT-Rv

GAGGGCAACGATGTCCTGATCAC

ZmPRX365-qRT-Fw

GAGATGACGACCGCTCCCATTG

ZmPRX365-qRT-Rv

AGCGGGCTTATGTTGCCCATC

ZmPRX648-qRT-Fw

TCCGCCTCCACTTCCATGACTG

ZmPRX648-qRT-Rv

ATCGCGTCGATCACCTCGTACC

ZmPRX731-qRT-Fw

CGGTGTTCGAGGTGATGGGCTAC

ZmPRX731-qRT-Rv

GCAGCAGTATGAGCGCCATGTTG

Figure S1. Model of the biosynthetic pathway of hydroxamate siderophores in Colletotrichum graminicola

L-ornithine L-ornithineN5-monoxygenase

Sid1

N5-hydroxyornithine N5-transacylase

CoA

glycine serine

Nps2

N5-acyl-N5hydroxyornithine nonribosomal peptide synthetases

i t intracellular ll l siderophores id h ferricrocin

acyl CoA

CoA

Nps6

secreted t d siderophores id h coprogen coprogen B α N -methylcoprogen B dimerumic acids

Fig. S1. The first step in the biosynthesis of hydroxamate siderophores in fungi is N5-hydroxylation of L-ornithine, catalyzed by the L-ornithine-N5-monooxygenase Sid1. In subsequent steps, the hydroxamate group is formed by N5acylation of N5-hydroxyornithine, and siderophore synthesis is completed by covalently y linking g the hydroxamate y residues byy nonribosomal p peptide p synthey tases. These enzymes require activation by 4'-phosphopantetheinylation, as catalyzed by the 4'-phosphopantetheinyl transferase Ppt1 . The nonribosomal peptide synthetases Nps6 is required for synthesis of secreted siderophores. The intracellular siderophore ferricrocin is synthesized by Nps2.

Figure S2. Phylogenetic tree of the putative siderophore biosynthetic enzymes Sid1 and Nps6 of C. graminicola 100

A Sid1

66

C. graminicola

C. higginsianum V. dahliae 100 V. V albo-atrum G. zeae M. oryzae N. crassa 9074 100 S. macrospora C. purpurea 100 M. anisopliae 100 M. acridum M. graminicola 82 84 P. nodorum A. otae CBS T. stipitatus 88 C. posadasii str. silveira P. chrysogenum A. terreus 99 A. nidulans 75 60 80 A. fumigatus 80 A. clavatus A. capsulatus 96 S. sclerotiorum 99 B. fuckeliana Pseudomonas sp. 80 B. multivorans 80 R. eutropha U. maydis S. reilianum Puccinia graminis f. sp. tritici

B

N. haematococca M. anisopliae T. virens T. reesei 100 N. crassa S. macrospora

73 100

Nps6

100 99 89 60

57

M. oryzae V. dahliae V. albo-atrum C. gloeosporioides C. higginsianum

C. graminicola C. posadasii C. immitis U reesii U. E. dermatitidis P. brasiliensis A. dermatitidis

100

99

55 97 95

A. capsulatus 100

91 100 88

G. fujikuroi G. zeae A. flavus A. clavatus A. fumigatus 55 100

A. alternata A. brassicicola C. sativus C. heterostrophus C. carbonum C. miyabeanus

0.05

0.1

Fig. g S2. Phylogenetic y g tree of Sid1 and Nps6 p of C. ggraminicola. (A) The phylogenetic tree indicates close relatedness of the ornithine N5-mono-oxygenase Sid1 of C. graminicola with other ornithine N5-monooxygenases of filamentous fungi. With the exception of Pyrenophora tritici-repentis (containing three monooxygenases), Coccidioides immitis and Coccidioides posadasii (con-taining two monooxygenases) all other fungi shown here contain one single mono-oxygenase. Sid 1 proteins of basidiomycetes show clear divergency to those of ascomycetes. Puccinia graminis f. sp. tritici shows very low similarity to ornithine N5-monooxygenases. (B) The phylogenetic tree indicates close relatedness of Nps6 of C. graminicola with other Nps6 proteins of filamentous fungi. A capsulatus, A. capsulatus Ajellomyces capsulatus; A. A dermatitidis, dermatitidis Ajellomyces dermatitidis; A. A alternata, alternata Alternaria alternata; A. A brassicicola, Alternaria brassicicola; A. otae, Arthroderma otae; A. clavatus, Aspergillus clavatus; A. fumigatus, Aspergillus fumigatus; A. flavus, Aspergillus flavus; A. nidulans, Aspergillus nidulans; A. terreus, Aspergillus terreus; B. carbonum, Bipolaris carbonum; B. oryzae, Bipolaris oryzae; B. sorokiniana, Bipolaris sorokiniana; B. fuckeliana, Botryotinia fuckeliana; C. purpurea, Claviceps purpurea; C. immitis, Coccidioides immitis; C. posadasii, Coccidioides posadasii, C. heterostrophus, Cochliobolus heterostrophus; C. graminicola, Colletotrichum graminicola; C. gloeosporioides, Colletotrichum gloeosporioides; C. higginsianum, Colletotrichum higginsianum; F. fujikuroi, Fusarium fujikuroi; F. graminearum, Fusarium graminearum; M. oryzae, Magnaporthe oryzae; M. acridum, Metarhizium acridum; M. anisopliae, Metarhizium anisopliae; M. graminicola, Mycosphaerella graminicola; N. haematococca, Nectria haematococca; N. crassa, Neurospora crassa; P. nodorum, Parastagonospora nodorum; P. chrysogenum , Penicillium chrysogenum; P. graminis f. sp. tritici, Puccinia graminis f. sp. tritici; P. tritici-repentis, Pyrenophora tritici-repentis; S. pombe, Schizosaccharomyces pombe; S. sclerotiorum, Sclerotinia sclerotiorum; S. macrospora, Sordaria macrospora; T. stipitatus, Talaromyces stipitatus; T. reesei, Trichoderma reesei; U.reesii, Uncinocarpus reesii; U. maydis, Ustilago maydis; V. alfalfa, Verticillium alfalfa; V. dahliae, Verticillium dahliae. Numbers above or below branches in A and B indicate percent of bootstrap support when bootstraps are >50% for each clade, performed with 1000 repetitions.

Figure S3. Construction of the SID1:eGFP and PSID1:eGFP cassettes F1

A

TTUBB

POLIC

nat1

PSID1

SID1

TTRPC

eGFP

R1 3.4 kb

kb V ∆sid1 1 * 3.4

B

Δsid1 + SID1:eGFP 3 4 5 6 7 8 9 10 11 12 * * * * * *

2 *

F1

C

TTUBB

POLIC

nat1

PSID1

TTRPC

eGFP

R1

1.6 kb

kb V ∆sid1 1 * 1.6

D

Δsid1 + PSID1:eGFP 2 3 4 5 6 7 * * * *

F

E

WT

∆sid1

Δsid1 +

SID1:eGFP

C 2 CgM2

Δsid1

Δsid1 +

SID1:eGFP

WT

8 *

Δsid1 +

co

co

co

PSID1:eGFP

ap

hp

ap

100

H

n = 2030

80 60 40

*

20

*

0

*

Infection structurees (%)

G

WT

Δsid1

Δsid1 +

SID1:eGFP

WT

Δsid1

Δsid1 +

WT+

SID1:eGFP SID1:eGFP

Fi S3. Fig. S3 Construction C i off the h SID1:eGFP SID1 GFP andd PSID1:eGFP GFP cassettes (A and C) Structure of the SID1:eGFP and PSID1:eGFP constructs. SID1, SID1 gene of C. graminicola; PSID1, SID1 promoter; eGFP, enhanced GFP gene; TTRPC, TRPC terminator from A. nidulans; nat1, nourseothricin acetyltransferase gene from Streptomyces noursei; POLIC, OLIC promoter from A. nidulans; TTUB1, TUB1 terminator of Botrytis cinerea. F1 and R1, SID1-KO-nest-Fw.1 and EGFP-Rv.2 primers. 3.4 kb and 1.6 kb are the expected size of the correct integration sizes of SID1:eGFP and PSID1:eGFP constructs, respectively. (B and D) PCR products confirming the correct integration size of the SID1:eGFP and PSID1:eGFP constructs, respectively. Lanes marked with asterisks indicate expected construct sizes. ((E)) The SID1:eGFP construct functionallyy complemented p the Δsid1 strain and fullyy restored wild-type yp ggrowth and formation of conidia (Δsid1 + SID1:eGFP), the PSID1:eGFP construct did not (Δsid1 + PSID1:eGFP). (F) Microscopical inspection of C. graminicola WT, Δsid1 and Δsid1+SID1:eGFP strains at 24 HPI; ap, appressoria; co, conidia; hp, hyphopodia. Bar = 10 μm. (G) Germination and appressorium formation rates of the WT, Δsid1 and Δsid1+SID1:eGFP strains at 24 HPI. ger, germination rate (white bars); nma, rate of formation of non-melanized appressoria (grey bars); ma, rate of formation of melanized appressoria (black bars); asterisks represent significance groups (p < 0.001, n =2030); error bars = + SD. (H) Virulence assay of the eGFP strains on intact leaf segments. Δsid1+SID1:eGFP and WT+SID1:eGFP strains had full virulence and were indistinguishable from the WT strain. Δsid1 strains were avirulent on intact leaf segments.

Figure S4. Generation of Δsid1 and ∆nps6 strains of C. graminicola by homologous recombination.

A

B 5´F

5´F

3´F

hph

hph

A

A SID1

5´F M

5´F

3´F M

5949 bp

NPS6 E

A

M

C

hph M

bp WT 1 5949 4837

A 5177 bp

E

E

probe

probe 5´F

NPS6

5´F

3´F 4837 bp

deletion strains 2 3 4 5 ect

E

M

D

hph A

NPS6 A 77054bp

disruption strains bp WT 1 2 3 4 5 6

E

7 ect

7054 5177

Fig. S4. Generation of Δsid1 and ∆nps6 strains of C. graminicola by homologous recombination. (A) Scheme of targeted deletion of SID1 by homologous recombination. The full ORF of the SID1 gene was replaced by a 1798-pb fragment carrying the hph cassette. (B) Scheme of targeted deletion of NPS6 by homologous recombination. A 2692-bp fragment carrying the hph gene (not to scale) replaced a 3229-bp fragment of the NPS6 gene. In A and B, 3´F and 5´F indicate right and left flanks; probe, probe used for the Southern blot analyses; A, AgeI; E, EcoRV; M, MunI restriction sites used for construction of the KO cassette y and for Southern blot analysis. (C) Southern blot analysis performed with MunI-digested genomic DNA from WT, ectopic (ect), and Δsid1 strains showed that the 4837-bp fragment had replaced the 5949-bp WT band in all independent Δsid1 strains. A transformant with an ectopically integrated deletion construct showed an extra band in addition to the 5949-bp WT band. (D) Southern blot analyses performed with EcoRV-digested genomic DNA of WT, ectopic (ect), and Δnps6 strains showed that the 5177-bp WT band had been replaced by a 7054-bp fragment in all independent Δnps6 strains. A transformant with an ectopically integrated deletion construct showed an extra band in addition to the 5177-bp WT band.

Figure S5. MS analyses of siderophores characterized in C. graminicola

Fig. off siderophores characterized Fi S5. S5 MS analyses l id h h i d in i C. C graminicola. l The compounds with an absorption at 430 nm shown in Fig. 2 were subjected to MS analysis as described in Materials and Methods. Siderophore-derived molecular masses are shown in bold. All ferric siderophores were detected in the protonated form (M-2H+Fe)+, coprogen and ferricrocin were additionally detected as the sodium adduct (M-3H+Fe+Na) +, and ferricrocin also as the potassium adduct (M-3H+Fe+K)+.

Figure S6. Conidiation of ∆sid1 and ∆nps6 strains on media containing different concentration of Fe(III)-EDTA

WT

ect. SID1

Δsid1

ect. NPS6

Δnps6

0

50 FeIII-EDTA

100 (µM)) (µ

Fig. S6. Conidiation of ∆sid1 and ∆nps6 strains on OMA agar containing different concentration of Fe(III)-EDTA. The addition of EDTA-chelated iron (Fe(III)-EDTA) partially (∆sid1) or fully (∆nps6) restored conidiation of the siderophore-deficient strains ∆sid1 and ∆nps6. Strains harboring ectopically integrated deletion cassettes (ect. SID1 d ect. SID1and t NPS6) were indistinguishable i di ti i h bl from f the th WT strain. t i

Figure S7. Morphological defects of conidia of ∆sid1 and ∆nps6 strains formed in liquid medium.

A

Δsid1

Δnps6

B

30

Length ((µm)

WT

a

a

20

b

b b

b b

10

c

0

WT ect.

Δsid1

Δnps6

Fig. S7. Morphological defects of conidia of ∆sid1 and ∆nps6 strains formed in liquid medium. (A) Morphology of conidia formed in complete liquid medium (CM). The shape of conidia of strains harboring an ectopically integrated deletion cassette was indistinguishable from that of the WT. Bar = 10 µm. (B) Quantitative measurement of length of conidia formed in CM medium. Different letters indicate significant differences between ∆sid1 and ∆nps6 strains and the WT and the ectopic (ect.) strain (P ≤ 0.001, n =2660). Bars represent + SD.

Figure S8. Conidiation defects of ∆sid1 and ∆nps6 strains on maize leaves

A

B 250

ect.

av Δsid1

av Δnps6

conidia / ml x 104

WT

a

200

a ab

150 100

b b

c

b

50 0

d Mock

WT

ect.

∆sid1

∆nps6

Mock

WT

ect.

∆sid1

∆nps6

av

non-wounded

wounded

Fig. S8. Conidiation of ∆sid1 and ∆nps6 strains on non-wounded and wounded leaves. (A) Asexual sporulation on wound-inoculated wound inoculated maize leaves at 6 DPI DPI. The WT and the ectopic (ect.) strains formed acervuli and produced conidia. The ∆sid1 and ∆nps6 strains showed defective acervuli (av), which produce severely reduced numbers of conidia. Bars are 10 µm. (B) Quantification of spores formed on non-wounded and wounded maize leaves after infection with the WT, an ectopic, ∆sid1 or ∆nps6 strain at 6 DPI. Different letters indicate significant differences (P ≤ 0.001, n = 436) Bars represent + SD.