Xin Liu et al., Curr Genet (2014)
Involvement of threonine deaminase FgIlv1 in isoleucine biosynthesis and full
virulence in Fusarium graminearum
Xin Liu· Jianhong Xu · Jian Wang · Fang Ji · Xianchao Yin · Jianrong Shi
Key Lab of Food Quality and Safety of Jiangsu Province-State Key Laboratory Breeding
Base/Key Laboratory of Control Technology and Standard for Agro-product Safety and
Quality (Nanjing), Ministry of Agriculture/Institute of Food Quality and Safety, Jiangsu
Academy of Agricultural Sciences, Nanjing, 210014, China
Correspondence: Jianrong Shi, e-mail: jianrong63@126.com
Xin Liu et al., Curr Genet (2014)
Supplementary figure captions
Fig. S1 Phylogenetic tree generated by using the neighbor-joining method with Mega
4.1 software on the basis of deduced amino acid sequences of FgIlv1 from Fusarium
graminearum strain PH-1 (indicated in the blank boxes), and those from Aspergillus
fumigatus (AfIlv1, EDP50192.1), A. nidulans (AnIlv1, CBF75287.1), Botrytis cinerea
(BcIlv1, CCD51125.1), Candida albicans (CaIlv1, EEQ46020.1), F. fujikuroi
(FfIlv1,CCT6299.1), F. oxysporum (FoIlv1, EMT61215.1), F. verticillioides (FvIlv1,
EWG36249.1), Magnaporthe oryzae (MoIlv1, ELQ39544.1), Neurospora crassa
(NcIlv1, XP_959505.1), Penicillium digitatum (PdIlv1, EKV04286.1),
Saccharomyces cerevisiae (ScIlv1, YER086W), and Sclerotinia sclerotiorum (SsIlv1,
XP_001586016.1).
Fig. S2 Generation and identification of FgILV1 gene deletion mutant. a Gene replacement
strategy for FgILV1 gene. The hygromycin resistance cassette (HPH) is denoted by the large
black arrow. Primer binding sites are indicated by arrows (see Table S1 for the primer
sequences). b Reverse transcription PCR analysis of FgILV1 expression in the wild-type
PH-1, ΔFgIlv1-3 and ΔFgIlv1-9C using cDNA as template. NC is a negative control without
template cDNA in the PCR amplification. c Southern blot hybridization analysis of the
wild-type PH-1, ΔFgIlv1-3 and ΔFgIlv1-9C using a 980-bp FgILV1 upstream fragment as a
probe. Genomic DNA of each strain was digested with Sac II.
Fig. S3 Generation and identification of FgCYS3 gene deletion mutant. a Gene replacement
strategy for FgCYS3 gene. The neomycin resistance cassette (NEO) is denoted by the large
gray arrow. Primer binding sites are indicated by arrows (see Table S1 for the primer
sequences). b Reverse transcription PCR analysis of FgCYS3 expression in the wild type
strain PH-1, ΔFgCys3-2 and ΔFgIlv1ΔFgCys3-10 using cDNA as template. NC is a negative
control without template cDNA in the PCR amplification. c Southern blot hybridization
analysis of the wild type strain PH-1, ΔFgCys3-2 and ΔFgIlv1ΔFgCys3-10 using a 838-bp
FgCYS3 upstream fragment as a probe. Genomic DNA preparation of each strain was
digested with EcoR I.
Xin Liu et al., Curr Genet (2014)
Fig. S1
Xin Liu et al., Curr Genet (2014)
Fig. S2
Xin Liu et al., Curr Genet (2014)
Fig. S3
Xin Liu et al., Curr Genet (2014)
Table S1. Oligonucleotide primers used in this study and their relevant characteristics.
Primer
Sequence(5’-3’)
Relevant characteristics
A1
ATggtaccATGCCACCAGCGCAAGAT
PCR primers for amplification of the
A2
ATctcgagAGGGTGTGAAGAGGTGAAAGA
upstream fragment of FgILV1.
A3
ATaagcttCTTGGTTTTGGCGTTTGGTT
PCR primers for amplification of the
A4
ATgagctcATTGACGCAGATGAGCCGA
downstream fragment of FgILV1.
A5
AATCCACAATCTACGTCACCC
PCR primers for the identification of
A6
CAACACACATACCCAAATTCG
FgILV1disruption mutants.
Ilv1-all-F
ATGCCTTCTGCTGATCCCA
PCR primers for amplification of full cDNA
Ilv1-all-R
TCATGATCGCAAAAATGTCTT
sequence of FgILV1.
Ilv1-com-F
ATggtaccAGACAGGAACTTTCAGAGGGC
PCR primers for amplification of the entire
Ilv1-com-R
ATaagcttAGACACTGGATCCTATTCGGG
FgILV1 gene including 901-bp the promoter
region and 508-bp terminator region.
Ilv1-RT-F
TGACCGAGTATAGCGCAAACC
PCR primers for the detection of FgILV1
Ilv1-RT-R
TTGAGGAGGACATTGCATTC
transcription.
B1
ATggtaccAATGGAATGGAATGAGCCGT
PCR primers for amplification of the
B2
ATctcgagAGTATAAACTCGGCGTTCCAG
upstream fragment of FgCYS3.
B3
ATaagcttTTTGGATAATTCCAATTGCG
PCR primers for amplification of the
B4
ATggatccTGCCCCGAAGTGTTCGTT
downstream fragment of FgCYS3.
B5
CAAAACAAATCACACCAGGC
PCR primers for the identification of FgCYS3
Xin Liu et al., Curr Genet (2014)
B6
AAGCATCCTCAGACAAAGGAA
disruption mutants.
Cys3-RT-F
GGAGCCGTGATCGAGTCTAT
PCR primers for analysis of FgCYS3
Cys3-RT-R
TCGGAGACGGAAATGACAT
expression.
Neo-F
GGAGGTCAACACATCAATGCT
PCR primers for amplification of NEO.
Neo-R
TCAGAAGAACTCGTCAAGAAG