In order to investigate the role of PacC in the growth of E. festucae and symbiosis with the host grass, pacC deletion (ΔpacC) and constitutive active (ΔpacC/pacCCA; henceforth called pacCCA) mutants were generated to give acid- and alkaline-mimicking mutations, respectively.
45 Figure 3.1. Multiple sequence alignment of E. festucae PacC with homologues from other fungi. Amino acid sequences from F. graminearum (ADO60821), M. oryzae (XP_003713788) and N. crassa (XP_957214) are shown in the alignment. The zinc finger regions are shaded in grey and the three interacting regions of the PacC protein; regions A, B and C are coloured in red,
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blue and purple, respectively. The signalling protease box is highlighted in yellow, and the red arrow marks the arginine residue, the codon for which was mutated into a stop codon in the pacCCA mutant.
The ΔpacC mutant was generated by transforming wild-type E. festucae protoplasts with a PCR-amplified fragment from the ΔpacC replacement construct, pYL1 (Section 2.8.1; Appendix 6.2.1). An initial PCR screen identified 12 out of 96 geneticin-resistant (GenR) transformants with patterns consistent with targeted replacement of the pacC locus (Figure 3.2A and B). A second PCR screen of 10 ΔpacC transformants further excluded transformant #29 from analysis (Figure 3.2C and D). Subsequently, Southern blot analysis was performed with NdeI/PstI-digested genomic DNA from 5 ΔpacC transformants and probed with a DIG-labelled linear fragment amplified from pYL1. The results revealed that all 5 mutants had ‘clean’ single-copy replacement events (Figure 3.3).
The strategy employed to generate the constitutive active PacC mutant was to transform the ΔpacC mutant with a truncated version of the pacC gene (pacCCA), lacking the ‘C’ interacting region of the protein (Tilburn et al., 1995). The translated protein is expected to be in an ‘open’ state, accessible for cleavage by the processing protease and as a result activated independent of a pH-signal. To achieve this, the CGA codon coding for arginine 464 of the E. festucae PacC protein was altered to a stop codon (TGA) by site-directed PCR mutagenesis. To bypass the need for splicing of the transcript, the construct was also generated from the pacC cDNA sequence. The resulting pacCCA sequence was placed under the control of the Ptef promoter and inserted into a backbone vector to make the pacCCA vector, pYL3 (Appendix 6.2.2; section 2.8.3). Transformation of ΔpacC mutant #8 with pYL3 resulted in 24 geneticin and hygromycin-resistant (GenRHygR) transformants. An initial PCR screen was performed to interrogate the flanking regions of the integrated construct (Figure 3.4A to C) followed by another PCR screen to check the integrity of each element of the pacCCA construct (Figure 3.4D to G). Results of the PCR screens revealed 8 transformants with patterns indicative of intact constructs (Figure 3.4). qRT-PCR analysis was subsequently performed using total RNA extracted from mycelia and showed that expression of the pacCCA construct was variable among these 8 transformants, with pacCCA #14 showing the highest expression level (Figure 3.5). Based on this result, pacCCA #14 was selected for further experiments to test the
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phenotype of the pacCCAmutant. For other experiments, two additional pacCCA mutants expressing high levels of the pacCCAconstruct; #19 and #22 were included.
Figure 3.2. PCR screens of ΔpacC transformants. (A) Strategy of ΔpacC first PCR screen using primer pairs YL15/YL16 that generates a 2.6 kb fragment in wild-type, 2.1 kb fragment in ΔpacC and both fragments in ectopic mutants. (B) Gel showing bands amplified from 96 ΔpacC transformants using primers YL15/YL16. P: positive plasmid control pYL1. ΔpacC strains of interest; #8, 11, 37, 73 and 96 are indicated. (C) Strategy of ΔpacC second PCR screen using four
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primer pairs; YL20F/YL20R and YL21F/ YL21R generate 2.2 kb and 2.1 kb fragments, respectively, in wild-type but none in ΔpacC transformants; YL20F/YL22R and YL22F/YL21R give 2.2 kb and 2.1 kb fragments in ΔpacC but none in wild-type. (D) Gel showing bands amplified from 10 ΔpacC transformants with primer pairs YL20F/YL20R (upper left panel), YL21F/YL21R (bottom left panel), YL20F/YL22R (upper right panel) and YL22F/YL21R (bottom right panel).
Figure 3.3. NBT/BCIP-stained Southern blot analysis of wild-type and ΔpacC transformants.
(A) Schematics of wild-type pacC genomic locus; PCR-amplified linear transformation fragment from pYL1; and the nptII-replaced pacC locus in the ΔpacC mutant. Grey regions depict regions for double cross-over recombination events between the homologous sequences (in yellow). The cleavage sites of NdeI and PstI restriction enzymes used in the genomic digest are indicated. A putative and uncharacterised gene downstream of pacC, EfM2.068170, as predicted by the MAKER gene prediction tool is indicated in the figure. (B) Southern blot of NdeI/PstI digested genomic DNA from wild-type and ΔpacC transformants, probed with DIG-dUTP-labelled linear fragment from pYL1 and visualised with NBT/BCIP.
49 Figure 3.4. PCR screens of pacCCA transformants. (A) Strategy of first PCR screen of pacCCA transformants using primer pairs YL25F/YL25R and YL26F/YL26R that generate 1.7 kb and 2.0 kb fragments in pacCCA transformants, respectively. (B and C) Gel of first PCR screen of 24 pacCCA transformants using primer pairs YL25F/YL25R (B) and YL26F/YL26R (C). P: positive plasmid control pYL3. pacCCA strains #1, 2, 6, 13, 14, 19 and 22 are indicated. (D) Strategy of second PCR screen of pacCCA transformants using primer pairs YL24F/YL24R, YL9/YL10 and YL13/YL14 that give 0.8 kb, 1.4 kb and 0.6 kb fragments in pacCCA transformants, respectively. (E to G) Gels of second PCR screen of 22 pacCCA transformants with primer pairs YL24F/YL24R (E), YL9/YL10 (F) and YL13/YL14 (G). Δ: negative control using genomic DNA from ΔpacC as template. P: positive plasmid control pYL3. pacCCA strains #1, 2, 6, 13, 14, 19 and 22 are indicated.
50 Figure 3.5. Expression of pacCCA transcript in eight pacCCA transformants. Mycelia were
grown in 2.4% potato dextrose media for 3 days at 22°C and total RNA extracted from mycelia was used to synthesise cDNA and used for qRT-PCR analysis. Values were quantified by the 2(ΔCp) method normalised to the expression levels of the EF-2 reference gene. Data are representative of two technical replicates from a single biological replicate; Y-axis represents fold mRNA abundance to the EF-2 gene, error bars represent standard deviation from two technical replicates.