A novel mitochondrial membrane protein, Ohmm, limits fungal oxidative stress
resistance and virulence in the insect fungal pathogen Beauveria bassiana
Zhangjiang He2, Suhong Zhang2, Nemat O. Keyhani3, Yulin Song2, Shuaishuai
Huang2, Yan Pei2, Yongjun Zhang1, 2*
Supporting Information
Figure S1. Molecular manipulations of Ohmm; targeted gene disruption,
complementation, and overexpression. (A) Ohmm locus and gene replacement vector
p∆Ohmm. p∆Ohmm contains bar cassette flanked by 3' and 5' border sequences of
Ohmm. Homologous recombination (cross over event marked by “X”) resulting in a
region of Ohmm was replaced by the bar cassette. Arrow indicates the orientation of
transcription in the ORF. (B) Ohmm complementation vector; pCB-Ohmm,
containing the sulfonylurea resistance marker (sur). (C) Ohmm overexpression vector.
The vector contains the Ohmm-coding region under the control of the fungal
constitutive promoter PgpdA from Aspergillus nidulans. (D) PCR analysis of wild-type
strain (WT), ΔOhmm, p∆Ohmm ectopic integrant (Control), and Ohmm::Ohmm
transformant (complemented mutant). Desired integration events were confirmed by
PCR using primers S1 and S2. (E) RT-PCR confirmation of loss of gene expression in
the ΔOhmm disruption mutant and recovery in the complemented strain
(ΔOhmm::ΔOhmm). Total RNA was isolated as described in the Methods section.
Expression of Ohmm was examined using actin as the reference gene in wild type
(WT), ΔOhmm, p∆Ohmm ectopic integrant, and ΔOhmm::Ohmm transformants. (F)
Real-time RT-PCR determination of Ohmm transcript levels in the wild type (WT)
and Ohmm overexpression (OE-Ohmm) strains; actin and β-tubulin were used as
reference genes. Error bars indicate standard deviations. (G) Southern blot analysis of
p∆Ohmm
ectopic integration transformant
(control),
ΔOhmm,
and
Ohmm
overexpression (OE-Ohmm) strains. Genomic DNAs were digested with EcoRI and
probed using the bar gene as described in the Methods.
Figure S2. Gene disruption of Ohmm in the Hog1 strain. (A) Ohmm locus in the
ΔHog1 strain and gene replacement vector p∆Ohmm. The p∆Ohmm contains the sur
cassette flanked by border sequence of Ohmm. Homologous recombination (cross
over event marked by “X”) resulting in a region of Ohmm replaced by the sur cassette.
Arrow indicates the orientation of transcription in the ORF. (B) PCR analysis of
ΔHog1 strain and the double ΔHog1ΔOhmm mutan. The desired integration event
was confirmed by PCR using primers S1 and S2 and the integrity of the ΔHog1 strain
was verified by PCR using HS1 and HS2 (4861 bp, Hog1 homologous recombination
cassette containing the bar resistance marker). (C) RT-PCR confirmation of loss of
Ohmm gene expression in the ΔHog1ΔOhmm mutant. Total RNA was isolated as
described in the Methods section. Expression of Ohmm was examined using actin as
the reference gene in Bbhog1 mutant (Δhog1) and the double Hog1Ohmm mutant
strain. (D) Southern blot analysis of the double Hog1Ohmm mutant. Genomic
DNAs were digested with EcoRI and probed using partial fragment of the sur gene as
described in the Methods.
Table S1. mRNA size and DNA sequence analysis of SSH cDNA clones expressed in the WT strain but
with markedly decreased expression in the Hog1 mutant under osmotic stress for 24 h
Clone (GenBank
accession No.)
Size
(bp)
E value
H64
226
9.00E-42
H77
251
H231
Identity
(%)
Accession no.
Homologous sequence in GenBank
97
EJP66004
Beauveria bassiana AMP-binding enzyme
1.00E-08
92
XP_002413831
Ixodes scapularis proteasome subunit alpha type
516
5.00E-36
100
EJP63790
Beauveria bassiana aldo/keto reductase
H388
435
8.00E-75
97
EJP66797
Beauveria bassiana short-chain dehydrogenase
H55
511
7.00E-21
43
XP_003855870
Zymoseptoria tritici hypothetical protein
H59
553
5.3
30
ZP_09618764
H128
594
8.00E-53
89
EJP68629
H209
309
2.00E-62
100
EJP66466
H669
768
1.00E-50
86
EJP66533
H83
280
3.00E-15
97
EJP64424
H350
279
2.00E-07
100
EJP69867
H232
413
unknown
H235
205
unknown
H464
206
unknown
Legionella drancourtii hypothetical protein
LDG_5091
Beauveria bassiana hypothetical protein
BBA_02631
Beauveria bassiana hypothetical protein
BBA_04406
Beauveria bassiana hypothetical protein
BBA_04473
Beauveria bassiana hypothetical protein
BBA_06418
Beauveria bassiana hypothetical protein
BBA_00736
Table S2. Oligonucleotides used in this study
Paired primers
Sequence (5'–3')*
Remarks
YADE-based PCR walking
Y5P1/Y5P2
AGACTGTGCTCAACGCCATG/ AGGACCTGGGCATGATTCTC
Cloning 5' fragment of Ohmm
Y3P1/Y3P2
ATGGCGTTGAGCACAGTCTT/ATGAACCGGTGCGACTGCTC
Cloning 3' fragment of Ohmm
Cloning cDNA of Ohmm using 3' RACE
RaF1
CATGATGGCGACCTCTATCA
RaF2
TAATCCTTGGACGCTGCGAC
RaR1
TTAGCTATCCCAAATAGCT
RaR1
CAAGAGCCTTCTTGTCCGTC
Cloning FAD/NAD(P)-binding region
FD1/FD2
CGGAATTCCGATGGCGGCCGTTCTCTCCTT
/ATAAGAATGCGGCCGCTAAACTATTTAGCTATCCCAAATAGCTCCAT
GFP::Ohmm fusion vector
GF1/GF2
CGGAATTCCGATGGTGAGCAAGGGCGAGGA/
GACTTGTACAGCTCGTCCAT
Cloning of eGFP
OG1/OG2
ATGGACGAGCTGTACAAGTGGCTCGGGCATGATGGCGACCTCTATCAT/
GCGATATCCTATTAGCTATCCCAAATAGCT
Cloning of Ohmm containing
upstream sequence
Ohmm disruption vector p∆Ohmm construction and screen or confirmation of disruption strain
L1/L2
CCCAAGCTTATCATGATGGCGACCTCTAT
/TCAATGTCATCTTCTGTCGACATGACGAGGGTGCCACCATT
Cloning of 5'-end of Ohmm
R1/R2
TGCCCGTCACCGAGATCTAATAGTCAATACCATGAGCAGACAT
/GCTCTAGACGTTGAGAAGCACCACCACGGA
Cloning of 3'-end of Ohmm
B1/B2
GTCGACAGAAGATGACATTGA/CTATTAGATCTCGGTGACGGGCA
Cloning of bar cassette
S1/S2
ATCTCTCTCAAGATTCTCAT/TACACATTGTTATTAGGAGT
Screen or confirmation
disruption strain
of
Cloning Ohmm sequence used for construction of reverse complement vector pCB-Ohmm
RC1/RC2
CCTACGTATACAGCTGCAATGATTGGCT
/GCTCTAGATCCTTGTCTGGTAGGCGTAG
Cloning Ohmm coding region used for construction of overexpression vector
O1/O2
GGAATTCAAGACGTATATATGGTGCGA
/GCGATATCATTTAGCTATCCCAAATAGCT
Construction of Ohmm disruption vector used for disruption of the gene in ∆Hog1 mutant
L3/L4
R3/R2
SuF/SuR
GGAATTCATCATGATGGCGACCTCTAT
/TCAATGTCATCTTCTGTCGACATGACGAGGGTGCCACCATT
Cloning of 5'-end of Ohmm
CGGGAATTGCATGCTCTCACTCAATACCATGAGCAGACAT
/GACTAGTCGTTGAGAAGCACCACCACGGA
Cloning of 3'-end of Ohmm
GTCGACAGAAGATGACATTGA/GTGAGAGCATGCAATTCCCG
Cloning sur cassette
Confirmation of Hog1 disruption strain (∆Hog1)
HS1/HS2
GAATTCATTCACCCTTTGCGT/AAGTAATACCCTTTCAGAGCA
RT-PCR and real-time RT-PCR
aF/aR
βtF/βtR
TTGGTGCGAAACTTCAGCGTCTAGTC
/TCCAGCAAATGTGGATCTCCAAGCAG
TACTCTACGATTCGTCAAGT/TGCTGGAACAGAGCCGTCTT
ohF/ohR
ATCGCGACGCTCTTTAACCG/GCTATCCCAAATAGCTCCAT
FluG1/2
CCTCCCTAGTTTGGTCGCTTTCTC / CGCTGTCGGTAATCTGCTCCTC
FlbA1/2
CCAATCCACTCGCCGCTCTC / CGGAGGAAAGAGAATCGGTAGAGG
RT-PCR and qRT-PCR analysis
of actin
RT-PCR and qRT-PCR analysis
of β-tubulin
RT-PCR and qRT-PCR analysis
of BbohmM
RT-PCR and qRT-PCR analysis
of FluG
RT-PCR and qRT-PCR analysis
of FlbA
FlbB1/2
GCACTGACACGCCGACAAGAGC / CCGCCGCCGAAGCCTGTTG
FlbC1/2
TCCATCTCCAACTTGCTGGGTCTC / GGCGGCGTAGGCGGAAGG
FlbD1/2
CGGCAAGCGATGGGCAGAGATTG /ACGAGCAAGGTGACGGTAGAGGTG
hyd1F/R
ATCTACTGCTGCAACGAGAA/TACTGGATAAGACTGCCAAT
hyd2 F/R
AGTGTCAAGACTGGCGACAT/ATCCGAGGACGGTGATGGGA
RT-PCR and qRT-PCR analysis
of hyd2
CatB-F/R
AAGGCCATTCCCGTCATCAT/ AGCTCGCGGTCCCAGCATCT
RT-PCR and qRT-PCR analysis
of CatB
CatA-F/R
AGAGCAACGGCAAGCGTGTA / AGAAGTCGCTGCTCAGGGTA
CatC-F/R
CatP-F/R
GAGGAGCCCAGCAACGCACAAGAG/
CTGAGGACGACAAGGCCGCCATTC
GCTGGGCTGATCTGCTGGTCCTTG / TCCTTGCTGTAACGGTGGCTGTCG
CatD-F/R
CGGCTGCGGTGTCTTGTCCATAC / CCTTGTCGGCGTTCTGGCGAAG
Sod1-F/R
TCCACATCCACACCTTTGGT / AGGTCCAGCGTTGCCAGTCT
Sod2-F/R
AGCTCCTCGCCGCCATCACCA / AGCCGTCTTCCAGTTGATGA
Sod3-F/R
ATGACGCCAAGGCTGCTGCT / ATCAATGCCCAGTAGAGGCT
Sod4-F/R
CGAGATGGTCCTTACGGCTTCAG / GCTCCCAGGTGTTGAGGCATAG
HPX1-F/R
GCCGCCGCTTCTACTCGTCTG / CGTTGCTGCCGCCAGTACCG
Trx1-F/R
GCCATTTCGCCCTTCTTCACCAAG / CGCAGCCGCCGTTGTTGTAG
RT-PCR and qRT-PCR analysis
of CatA
RT-PCR and qRT-PCR analysis
of CatC
RT-PCR and qRT-PCR analysis
of CatP
RT-PCR and qRT-PCR analysis
of CatD
RT-PCR and qRT-PCR analysis
of Sod1
RT-PCR and qRT-PCR analysis
of Sod2
RT-PCR and qRT-PCR analysis
of Sod3
RT-PCR and qRT-PCR analysis
of Sod4
RT-PCR and qRT-PCR analysis
of HPX1
RT-PCR and qRT-PCR analysis
of trx1
*The enzyme sites are italicized.
RT-PCR and qRT-PCR analysis
of FlbB
RT-PCR and qRT-PCR analysis
of FlbC
RT-PCR and qRT-PCR analysis
of FlbD
RT-PCR and qRT-PCR analysis
of hyd1