Genomics and Applied Biology 2015, Vol. 6, No. 7, 1-8
9
Supplementary Materials
Supplementary Figure 1: PCR analysis for double mutant confirmation.
The Δ
trm-9
Δ
nca-2
double
mutants were verified by using the forward primers NCU04898-5F 5’
GGTTAGTGAGCTTTGAGTCG
3’
and NCU04736 -5F 5’
TACACTGGTAATGGACCACG 3’
specific for upstream of the open reading frame
of genes
trm-9
and
nca-2
, respectively, and with the common reverse primer 5HPHR 5’
ATCCACTTAACGTTACTGAAATC
3’ that is specific for the
hph
cassette used to generate the knockout
mutants (Colot et al., 2006; Deka et al., 2011). Amplification of PCR products of size
∼
1.2 and ~1.018 kb
indicate the presence
trm-9
and
nca-2
knockout alleles respectively. The wild-type was used as negative
controls for the knockout alleles (indicated in the parenthesis) using the allele specific primer pairs.
PCR
products were visualized in a 0.8% agarose gel with 1 kb DNA ladder.
Supplementary Figure 2:
Conidial cell count of wild-type,
trm-9,
nca-2,
and
trm-9
nca-2
double
mutant strains grown in Vogel’s glucose medium. Conidia count of wild-type and mutant strains are plotted
with relative counting with respect to wild-type. Error bars indicate the standard errors calculated from the
data for three independent experiments. Conidial cell count of
trm-9
nca-2
double mutant strain was less
than the parental single knockout mutant strains and wild-type
.
Supplementary Figure 3: Sequence alignment of
cmd
and
trm9
homologues.
(A) Sequence alignment
of the
Neurospora crassa
CaM homologues. (B) Sequence alignment of the
trm-9
homologues.
Conserved amino acids are indicated in black (100%), dark gray (>80%) and light gray (>60%). The
homologue sequences used for the sequence analysis are from
Ajellomyces capsulatus
(AC),
Ajellomyces
dermatitidis SLH14081
(AD),
Aspergillus fumigatus
(AF),
Aspergillus nidulans
(AN),
Botryotinia
fuckeliana B05.10
(BF),
Candida albicans
(CA),
Coccidioides immitis
(CI),
Cordyceps militaris CM01
(CM),
Coccidioides posadasii
(CP),
Dichotomomy cescejpii
(DC),
Esox lucius
(EL),
Grosmannia
clavigera kw1407
(GC),
Glomerella graminicola
(GG),
Gibberella zeae PH-1
(GZ),
Homo sapiens
(HS),
Komagataella pastoris
(KP),
Magnaporthe grisea
(MG),
Neurospora crassa
(NC),
Neurospora
tetrasperma
(NT),
Procambarus clarkii
(PC),
Phytophthora infestans T30-4
(PI),
Rhodomonas sp.
CCMP768
(RS),
Saccharomyces cerevisiae
(SC),
Schistosoma mansoni
(SM),
Spathaspora
passalidarum
(SP),
Trichoderma reesei
(TR), and
Talaromyces stipitatus ATCC 10500
(TS).
Supplementary Figure 4: Phylogenetic analysis of the (A) CaM and (B) TRM-9 proteins.
Protein
sequences are described using GenBank accession numbers, phylum is indicated at major clades, and bar
indicates scale of genetic distances. The homologue sequences used for the sequence analysis are from
Ajellomyces capsulatus
(AC),
Ajellomyces dermatitidis SLH14081
(AD),
Aspergillus fumigatus
(AF),
Arthroderma gypseum
(AG),
Aspergillus kawachii
(AK),
Aspergillus nidulans
(AN),
Aspergillus oryzae
(AO),
Botryotinia fuckeliana B05.10
(BF),
Candida albicans
(CA),
Coccidioides immitis
(CI),
Cordyceps
militaris CM01
(CM),
Coccidioides posadasii
(CP),
Dichotomomy cescejpii
(DC),
Drosophila
melanogaster
(DM),
Exophiala dermatitidis
(ED),
Esox lucius
(EL),
Eurotium rubrum
(ER),
Emericella
unguis
(EU),
Grosmannia clavigera kw1407
(GC),
Glomerella graminicola
(GG),
Gibberella zeae PH-1
(GZ),
Homo sapiens
(HS),
Komagataella pastoris
(KP),
Metarhizium anisopliae
(MA),
Magnaporthe
grisea
(MG),
Neurospora crassa
(NC),
Neurospora tetrasperma
(NT),
Ogataeapara polymorpha
(OP),
Paracoccidioides brasiliensis
(PB),
Procambarus clarkii
(PC),
Puccinia graminis f. sp. Tritici
(PG),
Phytophthora infestans T30-4
(PI),
Penicillium rolfsii
(PR),
Pyrenophora tritici-repentis
(PT),
Rhodomonas sp. CCMP768
(RS),
Saccharomyces cerevisiae
(SC),
Saccharomyces cerevisiae RM11-1a
(SC RM11),
Saccharomyces cerevisiae
x
Saccharomyces kudriavzevii VIN7
(SCSK),
Sordaria macrospora
(SM),
Spathaspora passalidarum
(SP),
Scheffersomyce sstipitis
(SS),
Trichophyton equinum
(TE),
Trichoderma reesei
(TR),
Talaromyces stipitatus ATCC 10500
(TS), and
Verticillium albo-atrum
(VA).
