Genomics and Applied Biology 2015, Vol. 6, No. 7, 1-8
6
colony morphology with matty-like colony growth
(Figure 2B). In addition, the dry weight of the strains
followed the order wild-type>
nca-2>
trm-9
>
trm-9
nca-2
(Figure 2C). The
trm-9
nca-2
double mutant also showed sensitivity to CaCl
2,
reduced aerial hyphae development and ultraviolet
(UV) survival (Supplementary Figure 8). Therefore,
these results suggested that lack of both
trm-9
and
nca-2
result in impaired growth, hyphae development
and conidial development in
N. crassa.
Carotenoids accumulation
We also analyzed carotenoid accumulation in
N.
crassa
in the presence of TFP and CPZ to investigate
the role of CaM in carotenoid accumulation. The
carotenoid profile of the wild-type strain in presence
of the inhibitors followed the order wild-type > 10 µM
CPZ > 20 µM CPZ >10 µM TFP > 20 µM TFP
(Figure 3A). Therefore, these results indicate the CaM
protein might modulate carotenoid accumulation in
N.
crassa
. The difference of carotenoids accumulation
in presence of TFP and CPZ might be due to the
difference of mechanism of inhibition mediated by
TFP and CPZ. Furthermore, carotenoids accumulation
in the
trm-9
nca-2
double
mutant was lower than
either of the parental single mutant strains, and
reduced further on medium supplemented with high
concentrations of CaCl
2
(Figure 3B). In addition,
nca-2
mutant was unable to grow on medium
supplemented with 0.3 M CaCl
2
or more
(supplementray Figure
8A) and consequently, no
carotenoids was accumulated; however, accumulation
of carotenoids in the
trm-9
was similar to the
wild-type (Figure 3B). Therefore, these results suggest
that
nca-2
plays a role in carotenoids biosynthesis.
Lack of both nca-2 and trm-9 affect in acquisition
of induced thermotolerance
We studied the ability of the
trm-9
nca-2
double
mutant in acquisition of induced thermotolerance. The
trm-9
nca-2
double mutants showed decreased
survival in induced thermotolerance as compared to
parental single mutants. The survival in induced heat
shock temperature followed the order
nca-2
>
trm-9
>
trm-9
nca-2
>wild-type (Figure 4). Therefore,
lack of both
nca-2
and
trm-9
had a negative effect in
acquisition of induced thermotolerance.
Conclusions
CaM and its target proteins mediate diverse cellular
functions. The CaM antagonists TFP and CPZ affect
growth, aerial hyphae development, carotenoids
accumulation and sexual development in
N. crassa.
In
addition,
trm-9
mutant has a slow growth phenotype
and less dry weight content. Moreover, the
trm-
9
nca-2
double mutant showed a severe growth
defect, less carotenoid accumulation, reduced conidial
count, an increased sensitivity to CaCl
2
, and reduced
viability in acquisition of thermotolerance induced by
heat shock temperature. Thus, in this study, we have
shown that
cmd
,
trm-9
, and
nca-2
genes play an
important role in growth, pigmentation, and stress-
tolerance in
N. crassa
.
Acknowledgements
The Fungal Genetic Stock Center (FGSC) generously waived
charges for strains and race tube. The FGSC was supported by
National Science Foundation grant BIR-9222772. We thank
Upasana Sarma for performing some initial experiments. VL was
supported by a Research Fellowship from the Ministry of
Human Resource Development, Government of India. This
work was supported partially by grants, including BT/PR3635/
BCE/8/ 892/2012, from the Department of Biotechnology,
Government of India.
References
Altschul S.F., Gish W., Miller W., Myers E.W., et al., 1990, Basic local
alignment search tool, J Mol Biol, 215:403-410
Benito B., Garciadeblas B., et al., 2000, Molecular cloning of the calcium
and sodium ATPases in
Neurospora crassa
, Mol. Microbiol,
35:1079-1088
Blum J.J., Hayes A., Jamieson G.A. Jr., Vanaman T.C., 1980, Calmodulin
confers calcium sensitivity on ciliary dynein ATPase, J Cell Bio,
87:386-397
Borkovich K.A., Alex L.A., Yarden O., Freitag M., et al., 2004, Lessons
from the genome sequence of
Neurospora crassa
: tracing the path from
genomic blueprint to multicellular organism, Microbiol Mol Biol Rev,
68:1-108
Bowman B.J., Draskovic M., Freitag M., Bowman E.J., 2009, Structure and
distribution of organelles and cellular location of calcium transporters in
Neurospora crassa
, Eukaryot Cell, 8:1845-1855
Bowman B.J., Abreu S., Margolles-Clark E., Draskovic M., et al., 2011,
Role of four calcium transport proteins, encoded by
nca-1
,
nca-2
,
nca-3
, and
cax
, in maintaining intracellular calcium levels in
Neurospora crassa
, Eukaryot Cell, 10: 654-661
Capelli N., Tuinen V.D., Perez O.R., Arrighi J.F., Turian G., 1993, Molecular
cloning of a cDNA encoding CaM from
Neurospora crassa
, FEBS
321:63-68
Chung W.S., Lee S.H., Kim J.C., Heo W.D., Kim M.C., Park C.Y., Park H.C.,
