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.
