Genomics and Applied Biology 2015, Vol. 6, No. 7, 1-8
5
. The TRM-9
possesses one
conserved E
1
-E
2
ATPases domain, and one halo-acid
dehalogenase like hydrolase domain (Supplementary
Figure 3B). In addition, phylogenetic analysis
revealed that CaM and TRM-9
Figure 3 Analysis of carotenoids content. (A) Carotenoid
content of wild-type strain in the presence of CaM antagonist
TFP and CPZ.
Standard errors calculated from the data for
three independent experiments are shown using error bars.
TFP
act as negative regulator for carotenoid accumulation whereas
CPZ act as positive regulator during carotenoid accumulation.
(B)
Carotenoids accumulation of
wild-type,
trm-9,
nca-2,
and
trm-9
nca-2
double mutant strains
during Ca
2+
stress.
Carotenoids extracted from these strains
grown in Vogel’s
liquid medium without CaCl
2
, supplemented with various
concentrations of CaCl
2
. Carotenoids were extracted and
estimated in
µg carotenoids per g of dry weight. Error bars
show the standard errors calculated from the data for three
independent experiments. Statistically significant values are
indicated by asterisks, *P < 0.05
proteins are clustered with homologues from related
(Supplementary Figure 4). Promoter analysis
revealed important putative regulatory elements
involved in transcription of
cmd
and
trm-9
gene by
using MatInspector software (Supplementary Figure
5). The NCU04736 gene was previously shown to
encode
nca-2
(Bowman et al., 2009; Bowman et al.,
2011).
Figure 4 Thermotolerance measurement of wild-type,
trm-9,
nca-2,
and
trm-9
nca-2
double mutant strains in induced
(44°C) and uninduced (30°C) conditions.
Each data point
represents the mean of three independent experiments
The
cmd
,
trm-9
, and
nca-2
genes are involved in
growth
We used CaM antagonists, trifluoperazine (TFP) and
chlorpromazine (CPZ) to study the effect of CaM
inhibition on growth of wild-type strain of
N. crassa
.
We found that both TFP and CPZ inhibit growth, a
hyphal branching, and development of aerial hyphae
in
N. crassa
(Figure 1). Moreover, addition of TFP (10,
20, 40, 60, 80, and 100 µM) or CPZ (20, 40, 60, 80, 100
µM) in the synthetic crossing medium (SCM) causes a
defect in perithecia formation in
N. crassa
and results
in a sterile phenotype (data not shown). In addition,
the
cmd
transcript level was found to be decreased in
the presence of TFP and CPZ as revealed by the real
time PCR analysis (Supplementary Figure 6). These
results suggest that CaM play a role in vegetative
growth, hyphal development, and sexual development
in
N. crassa
.
The
trm-9
mutant displayed a slow growth
phenotype (Figure 2A). However, the slow growth
phenotype of the
trm-9
mutant was not due to a
defect in the ergosterol profile (Supplementary Figure 7)
and the growth of the
trm-9
mutant was not affected
by addition of various amounts of CaCl
2
, sucrose and
NaCl in the medium indicating that the
trm-9
mutant
is insensitive to these stress conditions (data not
shown). The growth defect was more severe in the
trm-9
nca-2
double mutant that showed distinct
