5.2 Results
5.2.6 The ncs-1 nca-2 double mutant was hypersensitive to Ca and UV stress
Figure 5.7: Effect of Ca2+ stress on the growth of wild-type, single and double mutant strains. Colony diameter (cm h-1) of the indicated N. crassa strains were measured at regular intervals and plotted against various concentrations of CaCl2. Standard errors calculated from the data for three independent experiments are shown using error bars.
Table 5.5 Average colony growth rate of the wild-type, single and double mutant strains at different concentrations of CaCl2 (M)
Strain Average colony growth rate (cm h-1) at different concentrations of CaCl2 (M)
0 0.2 0.3 0.4
Wild-type 0.359 ± 0.009 0.327 ± 0.009 0.288 ± 0.018 0.241 ± 0.013 ncs-1 0.289 ± 0.018 0.218 ± 0.017 0.122 ± 0.022 0.074 ± 0.029 nca-2 0.309 ± 0.028 0.239 ± 0.007 0.187 ± 0.024 0.095 ± 0.013 mid-1 0.216 ± 0.011 0.397 ± 0.010 0.385 ± 0.009 0.335 ± 0.016 ncs-1 nca-2 0.213 ± 0.007 0.153 ± 0.009 0.091 ± 0.012 0.05 ± 0.020 ncs-1 mid-1 0.187 ± 0.013 0.251 ± 0.019 0.156 ± 0.015 0.134 ± 0.013 mid-1 nca-2 0.227 ± 0.013 0.180 ± 0.018 0.046 ± 0.010 0.028 ± 0.000
Figure 5.8: Dose response curves showing relative UV-sensitivity of the wild-type, single and double mutant strains. Approximately 103 conidia of the wild-type, single and double mutantstrains were plated on Vogel’s sorbose agar medium (in petridishes of 150 mm diameter) and irradiated with various UV doses. Percent survival was obtained by dividing the number of colonies from plates irradiated with UV dose by the number of colonies on plates with no UV exposure (control).
Table 5.6 Relative UV-sensitivity of the wild-type, single and double mutant strains Strain Percent survival at indicated UV doses (J m-2)
0 50 100 150
Wild-type 100 87.3 ± 2.08 40 ± 0.577 12.6 ± 1.1577
ncs-1 100 56.9 ± 0.90 18.9 ± 3.9 1.5 ± 0.25
nca-2 100 56.3 ± 1.15 12.48 ± 2.18 1.62 ± 0.74
mid-1 100 53.6 ± 1.52 17 ± 0.5 1.3 ± 0.17
ncs-1 nca-2 100 69.3 ± 2.51 10.1 ± 2.22 0
ncs-1 mid-1 100 62.6 ± 0.57 15.3 ± 0.35 2.2 ± 0.2 mid-1 nca-2 100 76.5 ± 1.36 34.8 ± 2.84 3.36 ± 1.15
5.2.7 The ∆∆∆∆ncs-1∆∆∆∆nca-2, ∆∆∆∆ncs-1∆∆∆∆mid-1, and ∆∆∆∆mid-1∆∆∆∆nca-2 double mutants showed a decreased sensitivity to the respiratory by-product CO2 and produced conidial bands
The analysis of the knockout mutants for the genes ncs-1, mid-1, and nca-2 has suggested their involvement in stress tolerance. Therefore, I tested the hypothesis that these mutants, either alone or in combination, could also tolerate the stress generated due to the accumulation of the respiratory by-product CO2. The conidiation of the wild-type in a race tube is suppressed due to accumulation of CO2. However, conidiation persists despite the CO2 accumulation in a race tube in the band (bd) mutant ras-1bd that carries a T79I point mutation in ras-1; therefore, the ras-1bd mutant is used in circadian rhythm studies (Sargent and Kaltenborn 1972; Belden et al. 2007). The wild-type, ras-1bd mutant, single and double mutants were inoculated at one end of the race tubes and kept at 25ºC under light for 24 h.
After 24 h, the growth front was marked and race tubes were transferred to continuous darkness. After every 24 h, growth front was marked under red safe light (Table 5.7). I found that both ∆ncs-1∆mid-1 and ∆mid-1∆nca-2 double mutants produced distinct conidial bands with an increased period length of 25.41 ± 0.48 h and 23.96 ± 0.58 h, respectively, without introducing the ras-1bd allele (Figure 5.9 A). The ras-1bd control showed a period length of 22.48 ± 0.72. The ∆ncs-1∆nca-2 double mutant showed a less robust effect and produced distinct conidial bands up to 72 h (Figure 5.9 A). To test if the conidial bands were produced due to an increased level of reactive oxygen species (ROS), I supplemented race tube medium with antioxidant N-acetyl-L-cysteine (NAC) to deplete ROS. Conidial bands were suppressed in the double mutants on addition of 10 mM NAC (Figure 5.9 B); however, NAC at 30 mM caused growth arrest, indicating a toxic effect at high concentration (Figure 5.9C). In summary, ∆ncs-1∆nca-2, ∆ncs-1∆mid-1, and ∆mid-1∆nca-2 double mutants showed lesser sensitivity than their parental single mutants and the wild-type to the respiratory by-product CO2 and produced conidial bands, which was possibly due to an increased ROS level in these double mutants. Thus, the finding that ∆ncs-1∆nca-2, ∆ncs- 1∆mid-1, and ∆mid-1∆nca-2 double mutants produced distinct conidial bands under
conditions normally unfavourable for conidiation in race tube, suggested that the products of these genes synthetically act as negative regulators, possibly in a parallel pathway of
circadian regulated conidiation.
Figure 5.9: Circadian regulated conidiation. (A) The ∆ncs-1∆mid-1 and ∆mid-1∆nca-2 double mutants produced distinct conidial bands at regular intervals in race tubes. In the
∆ncs-1∆nca-2 double mutant, bands were distinct till 72 h. (B) Rhythmic conidiation patterns of the ∆ncs-1∆mid-1 and ∆mid-1∆nca-2 double mutants were suppressed in the medium supplemented with 10 mM NAC. (C) Growth defects of the N. crassa strains in the medium supplemented with 30 mM NAC.
Table 5.7 Apical growth and distance of consecutive conidial bands of the wild-type, single and double mutant strains in race tubes in absence of NAC
Strain Distance (cm) of the apical growth front from the inoculation point in
the indicated time interval (h;
n=3)
Distance (cm) of consecutive conidial bands from the inoculation point in
the indicated time interval (h; n=3)
24 48 72 96 120 24 -48 48 -72 72-96 96 -120 ras-1bd 2.6 6.4 10.2 14 17.3 4.2 7.6 11.1 14.8
2.5 5.8 9.1 12.3 16.3 4 7.1 10.3 13.5 2.9 6.4 9.7 13.2 17.6 4.4 7.5 10.5 13.6 Wild-type 5.5 13.9 22.6 32
6.5 15.7 26.4 37 5.6 15.7 25.4 33
∆ncs-1 3.3 9.3 17.5 24 32 4.8 11.3 18.3 25.4 32.9 4.4 12.3 20.4 27.8 37
∆nca-2 6.5 14.6 23.3 32.2 3.4 9.9 18.4 26.6 5.6 15.7 24.4 32.9
∆mid-1 2.7 5.9 8.7 11.8
2.4 5.4 8.2 11.3
∆ncs- 1∆nca-2
3.3 8.1 12.9 17.3 21.9 5.7 9.7 3.7 9.9 16.1 23 27 6.1 9.1 4.1 10.5 16.4 22.5 28.8 7 11.3
∆ncs- 1∆mid-1
2 5 7.9 11 14 3.2 6.4 9.4 12.4 2.3 5.9 9.6 13 16.8 5.5 9.3 12.7 16.6 2.2 5.2 7.9 11 14.7 4.8 8.1 10.9 14
∆nca- 2∆mid-1
2.5 7 11 14.9 19.4 5.7 9.7 13.7 18.5 2.8 7.2 11.2 15.1 19.4 6.1 9.1 13.8 18.2 2.7 7.7 11.8 15.8 19.8 7 11.3 15.1 19.5
Figure 5.10: Model showing the cellular role of NCS-1, MID-1, and NCA-2. MID-1 is involved in passive influx of Ca2+, and NCA-2 is involved in reducing [Ca2+]c. The NCS-1 also possibly assist reducing [Ca2+]c. Therefore, all these three Ca2+ signaling proteins are play a role in maintaining a constant [Ca2+]c level, which is about 100 nM. NCS-1, MID-1 and NCA-2 are also involved individually or synthetically in UV-induced DNA damage repair, regulation of ROS level and circadian regulated conidiation. In addition, NCS-1 and MID-1 are possibly involved in maintaining tip-high Ca2+ gradient necessary for hyphal growth. The location of shown Ca2+ signaling proteins are as per the available published data and question mark indicate localisation yet to be identified.
Extracellular
Intracellular
The ∆ncs-1 ∆nca-2, ∆ncs-1∆mid-1, and ∆mid-1∆nca-2 double mutants were generated by crossing single mutant strains of opposite mating type and phenotypes of the double mutants were studied to determine genetic interaction of the ncs-1, mid-1, and nca-2 genes in N. crassa. The ∆ncs-1 ∆nca-2 double mutant showed novel colony morphology (Figure 5.3).
Moreover, the ∆ncs-1 ∆nca-2 double mutant displayed reduced aerial hyphae, slow growth rate, reduced carotenoid accumulation, increased sensitivity to Ca2+ and UV stress, and decreased sensitivity to CO2 in race tube than either of the parental mutants (Figures 5.4-5.9).
These results consistently suggested for negative genetic interaction of ncs-1 with nca-2. The phenotype of the ∆ncs-1∆nca-2 double mutant was aggravated possibly due to simultaneous knockdown of the compensatory pathway. The ∆ncs-1∆mid-1 double mutant showed aerial hyphae like the ∆ncs-1 mutant (Figure 5.4). However, the ∆ncs-1∆mid-1 double mutant had lower growth rates, and more carotenoid accumulation than either of the parental single mutants (Figures 5.5, 5.6). Besides, the ∆ncs-1∆mid-1 double mutant displayed an
intermediate Ca2+ sensitivity, and similar UV sensitivity in comparison to the parental single mutants (Figures 5.7, 5.8). The ∆ncs-1∆mid-1 double mutant also produced conidial bands in race tube even in the presence of the accumulated CO2 that was suppressed when the medium was supplemented with NAC (Figure 5.9). The ∆mid-1∆nca-2 double mutant showed aerial hyphae like the ∆nca-2 mutant, growth rate similar to the ∆mid-1 mutant; however, its carotenoid accumulation was more than either of the parental single mutants (Figures 5.4- 5.6). Moreover, stimulation of the growth rate of the ∆mid-1 mutant on medium containing CaCl2 supplement was completely abolished in the ∆nca-2 background and ∆mid-1 ∆nca-2 double mutant was hypersensitive to Ca2+ (Figure 5.7). Besides, the ∆mid-1∆nca-2 double mutant was more tolerant to UV and less sensitive to CO2 accumulation in race tube than the single mutants (Figures 5.8, 5.9). These results suggested that unlike the case for ncs-1 with nca-2, the interactions of ncs-1 with mid-1 and mid-1 with nca-2 are complex. In addition, ncs-1 mutation did not show any effect in the knockout background of another Ca2+-signaling gene NCU02814 (prd-4) for growth, Ca2+ and UV stress, and period length, therefore, ruling out the hypothesis that Ca2+-signaling genes randomly interact or similar genetic interactions exists between any of the Ca2+-signaling gene pair.
The genetic interaction studies also suggested the involvement of ncs-1, mid-1, and nca-2 genes in carotenoid accumulation. I found that carotenoid accumulation was
consistently increased in presence of the ∆mid-1 mutation and reached a peak of about six-
increased in the ∆ncs-1 and ∆nca-2 mutants; however, surprisingly, carotenoid accumulation in the ∆ncs-1∆nca-2 double mutant was like the wild-type. In N. crassa, biochemical synthesis of carotenoid pigments involves sequential enzymatic conversion of isopentenyl pyrophosphate (IPP) to geranylgeranyl pyrophosphate (GGPP), GGPP to phytoene (a colorless 40-carbon compound), and phytoene to carotenoid pigments, catalyzed by the enzymes GGPP synthetase, phytoenesynthetase, and phytoene dehydrogenase, respectively (Harding et al. 1969; Porter and Spurgeon 1979). Accumulation of xanthophyll
neurosporaxanthin and variable amounts of precursor carotenoid cause characteristic orange pigmentation of conidia and mycelia in N. crassa (De Fabo et al. 1976; Harding and Turner 1981; Nelson et al. 1989; Carattoli et al. 1991; Barba-Ostria et al. 2011; Dı´az-Sa´nchez et al.
2011). The GGPP synthetase, phytoenesynthetase, and phytoene dehydrogenase are encoded by albino (al) genes al-3, al-2, and al-1, respectively, and these genes are regulated by the white collar-1 (wc-1) gene that mediates a blue light induction process (Harding and Turner 1981; Nelson et al. 1989). In addition to these previously identified regulators, this genetic interaction studies suggested involvement of Ca2+ signaling pathway in modulating
carotenoid biosynthesis.
The Ca2+ sensitivity phenotype of the ∆ncs-1 mutant was not fully rescued by the
∆mid-1 mutation like their homologues in S. pombe (Hamasaki-Katagiri and Ames 2010;
Figure 5.7). Moreover, ∆mid-1 mutation did not rescue the defect in aerial hyphae
development in the ∆ncs-1 mutant. This might indicate functional differences of NCS-1 and MID-1 homologues in N. crassa and S. pombe. The UV sensitivity studies revealed that, in addition to the previously identified ncs-1 gene, mid-1and nca-2 also play a role in UV- induced DNA damage repair process (Figure 5.8). Moreover, genetic interactions of mid-1 with nca-2 and ncs-1 with nca-2 affect the UV survival in a negative and positive manner, respectively, in N. crassa. In addition, ∆ncs-1∆nca-2, ∆ncs-1∆mid-1, and ∆mid-1∆nca-2 double mutants showed lesser sensitivity to the respiratory by-product CO2 and produced conidial bands, which was possibly due to an increased ROS level, unlike their parental single mutants and the wild-type (Figure 5.9; Table 5.7).
These results suggested that complex genetic interactions of ncs-1, mid-1, and nca-2 regulate multiple cell functions in N. crassa. A model summarizing the cell functions of NCS-1, MID-1 and NCA-2 are shown in figure 5.10. A part of this chapter was published in Journal of Genetics (Deka and Tamuli 2013).