I tested three homokaryotic transformants carrying the NCU04379 transgene fortheir ability to complement the growth, CaCl2 and UV sensitivity phenotypes of the NCU04379.2 mutant. The homokaryotic transformants were found to complement the slow growth (Figure 3.13, Table 3.8), CaCl2 sensitivity (Figure 3.14 A, B; Table 3.9), CaCl2 deprivation by EGTA (Figure 3.15) and UV sensitivity (Figure 3.16 A, B) phenotypes of the NCU04379.2 mutant.
Therefore, I concluded that NCU04379 gene plays a role in growth, Ca2+ stress tolerance and UV survival in N. crassa.
S. no. Primer Sequence (5 3 ) Reference/source
1. NCU04379-5F GCTCGAAAGTTTAGTCCTGG FGSC
2. NCU04379-3R CCCAGTAACGTCTCTTTTGC FGSC
3. 5HPHR ATCCACTTAACGTTACTGAAATC This study
4. 6NCU04379-F AGCTCGAGCGTGATAAGCTG This study
5. 7NCU04379-R AGGGCGGATACGATTGTCTC This study
6. NcTUB1f AAC TTA CAA GAT GGC AGA GC This study
7. NcTUB1r AAG GGG TCA CTA CAC TGA GGG This study
A B
C D
Figure 3.9:Cloning of NCU04379 fragment from the wild-type for complementation analysis. (A) The primer pairs NCU04379-5F and NCU04379-3R, used to amplify the NCU04379 fragment (~3.972 kb in size) from the wild-type, are indicated. The relative positions of the start (ATG) and the stop (TAA) codons and the EcoRI restriction site are shown. The distance of the EcoRI site, ~1.448 kb and ~2.528 kb, respectively, from the NCU04379-3R and NCU04379-5F primers are also shown. The size of the PCR amplified product is shown below. (B) Agarose gel electrophoresis of the NCU04379 fragment (~3.972 kb in size) amplified from the wild-type, resolved in lanes 2 to 6; lane 1, 1 kb DNA ladder (NEB). (C) Agarose gel electrophoresis of the plasmid isolated from E. coli DH5 transformants; lane 1, 1 kb DNA ladder (NEB); lane 2, pBARGEM7-1 vector (uncut); lane 3, the plasmid shift indicates the ligation of the insert in the pBARGEM7-1 vector, and designated as pRD-1; lane 4, another plasmid isolate did not show any plasmid shift. (D) Restriction digestion analysis of the pRD-1 construct. The pRD-1 construct was digested with EcoRI and BamHI restriction enzymes and resolved in 0.8% agarose gel; lane 1, 1 kb
DNA ladder (NEB); lane 2, EcoRI digestion releases two fragments of different sizes, ~1.448 kb and ~6 kb from the pRD-1 construct; Lane 3, BamHI digestion resulted in linearization of the pRD-1 construct of size 8.472 kb.
A B Figure 3.10: Confirmation of the ∆∆∆∆NCU04379.2 mutant and the pRD-1 transformants by Southern hybridization. (A) Schematic representation to illustrate the fragments generated in the Southern hybridization. Position of the Xmn1 restriction enzyme cut sites (X1, X2, X3 and X4) in the locus region of the NCU04379 gene in the wild-type (a) and corresponding region in the ∆NCU04379.2 mutant (b). The 3.972 kb NCU04379 fragment, PCR amplified using primers NCU04379-5F and NCU04379-3R, was used as probe to hybridize the Southern blot. The Xmn I enzyme has two cut sites (X2 and X3) within the probe region that result in three fragments of size 645,1860, and 2175 bp in the wild-type, the knockout has only one cut site within the probe region (X3) that results only two fragments of size 645 and 3856 bp. (B) Southern analysis to confirm the ∆NCU04379.2 mutant and pRD- 1 transformant. Genomic DNA of the ∆NCU04379.2 mutant (KO), the wild-type (WT), and the pRD-1 heterokaryotic transformant (T-1) were digested with Xmn I and probed with a 3972 bp NCU04379 fragment amplified from the wild-type using the primers NCU04379-5F and NCU04379-3R (Table 3.6, entries 1, 2). Three fragments of size 645,1860, and 2175 bp were appeared in the wild-type (WT) due to the presence of two cut sites within the probe, the knockout (KO) has only one cut site within the probe region and thus resulted in two fragments of size 645 and 3856 bp. The 2175 bp fragment, specific for the wild-type, is restored in the transformant (T-1).
A
B
Figure 3.11: Verification of the NCU04379.2 mutant allele in the strains transformed with pRD-1 construct. (A) Schematics of the NCU04379 gene in the wild-type (a) and the NCU04379.2 mutant allele(b) are shown relative to the location on the linkage group. The position of NCU04379-5F forward (Table 3.7, entry 1) and the 5HPHR reverse (Table 3.7, entry 3) primers are shown. The 5HPHR is specific for the hph cassette used to generate the
NCU04379.2 mutant (Colot et al. 2006). Primers NCU04379-5F forward and 5HPHR are used to amplify a specific PCR product of size ~1.131 kb to verify the NCU04379.2 mutant allele. (B) Verification of the ∆NCU04379.2 mutant allele in the homokaryotic transformants by PCR using primers NCU04379-5F and 5HPHR. PCR products were resolved in a 0.8%
agarose gel; lane 1, 1 kb DNA ladder (NEB); lane 2, PCR amplification verifying the presence of the knockout allele (~1.131 kb in size) in the NCU04379.2 mutant (control);
lanes 3, 4, and 5, PCR amplification to verify the NCU04379.2 mutant allele (~1.131 kb in size) in the homokaryotic transformants HoP-7, HoP-10, and HoP-6, respectively; lane 6, wild-type control (no PCR product as target sequence for the 5HPHR primer is absent in the wild-type).
Figure 3.12: Verification of the expression of NCU04379 gene in the pRD-1
homokaryotic transformant. RT-PCR using primers NCU04379-6F and NCU04379-7R (Table 3.6, entries 4, 5), and RNA isolated from the wild-type, ∆NCU04379.2 mutant and the homokaryotic transformant-10. Amplification of a ~529 bp fragment confirms the expression of the NCU04379 gene. The RT-PCR products were resolved in a 1.2% agarose gel; lane 1, 100 bp ladder (NEB); lane 2, NCU04379 expression in the wild-type (control); lane 3, NCU04379 expression the homokaryotic transformant-10; lane 4, ∆NCU04379.2 mutant (control); lanes 5, 6, and 7, -tubulin control expression (~651 bp in size) in the wild-type, homokaryotic transformant-10, and the ∆NCU04379.2 mutant, respectively, using primers NcTUB1f and NcTUB1r.
Figure 3.13: Complementation of the slow growth phenotype of the NCU04379.2 mutant. Agar plug of the wild-type, the ∆NCU04379.2 mutant and the homokaryotic
transformant wereinoculated at one end of the race tube and incubated at 30°C till the growth front reaches the other end of the race tube (72 h). The homokaryotic transformant carrying the NCU04379 transgene showed growth rate similar to the wild-type. Error bars indicate the standard errors calculated from the data for three independent experiments.
Table 3.8Apicalgrowth of the wild-type, NCU04379.2 mutant and homokaryotic transformant in race tube
Strain Distance (mm) in race tube at the indicated time interval
0 h 12 h 24 h 36 h 48 h 60 h 72 h
Wild type 0 25 ± 3 77 ± 2 129 ± 5 184 ± 1 237± 7 283 ± 11 NCU04379.2 0 19 ± 2 55 ± 3 96 ± 1 140 ± 2 182 ± 3 220 ± 1 Homokaryotic
transformant
0 24 ± 2 73 ± 2 118 ± 2 174 ± 3 223 ± 5 268 ± 8
A
B
Figure 3.14: Complementation of the calcium sensitivity phenotype of the NCU04379.2 mutant. (A) Conidia of the wild-type, the ∆NCU04379.2 mutant and the homokaryotic transformants were inoculated in the center of petridishes (100 mm diameter) containing Vogel’s glucose agar medium supplemented with indicated concentrations of CaCl2 and incubated at 30°C for 2 days. The homokaryotic transformants (T-1-6, T-1-7, T-1-10) carrying the NCU04379 transgene showed growth similar to the wild-type on medium supplemented with CaCl2. (B) Average colony growth rate of wild-type, the ∆NCU04379.2 mutant and the homokaryotic transformants were plotted against different concentrations of CaCl2. The homokaryotic transformants (T-1-6, T-1-7, T-1-10) carrying the NCU04379 transgene displayed growth rates similar to the wild-type. Error bars indicate the standard errors calculated from the data for three independent experiments.
Table 3.9 Average colony growth rate of the wild-type, NCU04379.2 mutant and homokaryotic transformant at various concentrations of CaCl2
Strain Average growth rate (cm/h) at various concentrations of CaCl2 (M)
0 0.2 0.3 0.4
Wild-type 0.339 ± 0.041 0.283 ± 0.018 0.228 ± 0.021 0.185 ± 0.022 NCU04379.2 0.279 ± 0.010 0.153 ± 0.026 0.084 ± 0.019 0.012 ± 0.007 Homokaryotic
transformant
0.312 ± 0.004 0.271 ± 0.01 0.213 ± 0.007 0.178 ± 0.021
Figure 3.15: Complementation of the effect of EGTA on the NCU04379.2 mutant.
Average colony growth rate of the wild-type, the ∆NCU04379.2 mutant and the
homokaryotic transformant strains were plotted against different concentrations of EGTA.
The homokaryotic transformants (T-1-6, T-1-7, T-1-10) carrying the NCU04379 transgene displayed growth rates similar to the wild-type on the medium supplemented with EGTA.
Error bars indicate the standard errors calculated from the data for three independent experiments.
A
B
Figure 3.16: Complementation of the UV sensitivity phenotype of the NCU04379.2 mutant. (A) Qualitative analysis of the UV sensitivity phenotype. Approximately 105, 104, 103, 102, 101 conidia of the wild-type, the ∆NCU04379.2 mutant, the upr-1 mutant, and the homokaryotic transformants (homokaryotic T-1-7and 10) were spotted from left to right on the Vogel’s sorbose agar plates, irradiated with the indicated UV doses, and grown at 30°C for 3 days in dark. The ∆NCU04379.2 mutant was more sensitive to UV irradiation than the wild-type, and the homokaryotic transformant strains (T-1-7and 10) carrying the NCU04379 transgene displayed UV sensitivity similar to the wild-type. (B) Quantitative analysis of UV sensitivity by calculating the percent survival of the colonies on irradiation to UV doses.
Approximately 103 conidia of the wild-type, the ∆NCU04379.2 mutant, the upr-1 mutant, and the homokaryotic transformants (Homokaryotic T-1-7and 10) were plated onVogel’s sorbose agar plate and irradiated with different doses of UV. Percent survival was obtained by dividing the number of colonies from plates irradiated with UV by the number of colonies on plates with no UV irradiation (control). The homokaryotic transformant carrying the
NCU04379 transgene displayed UV sensitivity similar to the wild-type. Each data point represents the mean of at least three indepented experiments.