2. Materials and Methods
3.4 Regulation of secondary metabolism in E festucae by pH and PacC
sterile distilled water and transferred into Blankenship media buffered at the indicated pH(s): (A) pH 8, (B) pH 6.5 and (C) pH 5. Bars represent standard error of the mean calculated from three biological replicates.
3.4
Regulation of secondary metabolism in E. festucae by pH and
PacC
As PacC is known to regulate secondary metabolism in several different fungi (Espeso et al., 1993; Keller et al., 1997; Merhej et al., 2011; Schmitt et al., 2011; Trushina et al.,
5.2 5.3 5.4 5.5 5.6
Media only WT ΔpacC ΔpacC/pacC pacC-CA
pH
C
6.60 6.65 6.70 6.75 6.80Media only WT ΔpacC ΔpacC/pacC pacC-CA
pH
B
7.5 7.6 7.7 7.8 7.9 8.0 8.1Media only WT ΔpacC ΔpacC/pacC pacC-CA
pH
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2013) the role of PacC in regulating expression of the three main classes of secondary metabolites in E. festucae was tested. To this end, seven genes; three from the LTM cluster (ltmE, ltmP and ltmM), three from the EAS cluster (easA, dmaW and lpsA), and a single gene, perA, which codes for a multi-domain protein responsible for peramine biosynthesis, were selected for representative analysis. Two 5’-GCCAAG-3’ PacC consensus binding sequences were found in the promoter region of ltmP, one was found in the promoter region of dmaW, and no PacC binding sequences were found in the 1 kb promoter regions of the remaining five genes.
Standard curves were generated for each gene and primer efficiencies were found to range between 1.86 to 1.96 (Appendix 6.4.1 and 6.4.2). qRT-PCR was subsequently performed on total RNA extracted from wild-type mycelia grown in PD media and the results showed that only two genes; lpsA, and perA, were above the detection limit (Cp value <35; results not shown). In comparison, culture in the Blankenship medium, which has been shown to induce the expression of lol genes in E. festucae strain E2368 (Blankenship et al., 2001) resulted in detectable levels of ltmE, easA, dmaW, lpsA and perA (described in the next section). The expression levels of ltmP and ltmM remained undetectable in this medium. Interestingly, the expression levels of lpsA and perA, which were detectable in PD medium, were not upregulated in the Blankenship medium. Taking these results into consideration, the Blankenship medium was used in subsequent experiments to analyse the expression of secondary metabolite genes.
3.4.1 Role of pH and PacC in regulating secondary metabolism in E. festucae
The role of PacC in regulating secondary metabolism in E. festucae was subsequently investigated by incubating wild-type, ΔpacC, ΔpacC/pacC and pacCCA mycelia in pH- defined Blankenship media. A pilot experiment was performed using wild-type strain and time points of 6, 12 and 24 hours of incubation in pH-defined Blankenship media. However, after 12 and 24 hours, the differences in the expression of secondary metabolite genes and pacC between the different pH conditions were less distinguishable from 6 hours (results not shown), perhaps due to neutralisation of the media (Section 3.3.10). The experiment was thus repeated with a shorter time point of 2 hours. In this experiment, data were normalised against the expression of the 40S ribosomal protein S22 (RPS22) and elongation factor-2 (EF-2) reference genes, and both
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gave very similar results in all analyses. Consequently, just the data normalised to the RPS22 reference gene are presented.
The expression of pacC was firstly considered, and found to be pH-dependent in the wild-type strain. The expression of pacC at pH 8 was 1.9-fold the expression in pH 6.5; and the expression at pH 6.5 was 1.8-fold the expression at pH 5 (Figure 3.25A). As anticipated, the pacC transcript was not detectable in the deletion mutant. The expression of pacCCA construct, which product is expected to be activated independent of alkaline pH was stable across all pH conditions in the pacCCA mutant at a level similar to the expression of pacC in the wild-type strain at pH 6.5 (Figure 3.25A).
Figure 3.25. Effect of pH and pacC mutations on secondary metabolite gene expression in culture. (A to F) Steady-state mRNA levels of secondary metabolite genes in wild-type, ΔpacC #8 and pacCCA #14 mutants of E. festucae grown in culture. The transcript analysed in the pacCCA mutant in (A) refers to the pacCCA transcript and not the endogenous pacC transcript. Mycelia
0 0.1 0.2 0.3 pH 5 pH 6.5 pH 8 Fo ld R P S22 expr ess io n
A
pacC
Wild-type ΔpacC pacC-CA
ND ND ND 0.000 0.005 0.010 0.015 0.020 pH 5 pH 6.5 pH 8
B
ltmE
0 0.002 0.004 0.006 pH 5 pH 6.5 pH 8C
easA
0 0.001 0.002 0.003 pH 5 pH 6.5 pH 8D
dmaW
0.000 0.005 0.010 0.015 0.020 pH 5 pH 6.5 pH 8E
lpsA
0 0.01 0.02 0.03 0.04 pH 5 pH 6.5 pH 8F
perA
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were cultured in Blankenship media buffered at pH 6.5 for 5 days, washed, and transferred to fresh Blankenship media buffered at pH 5, 6.5 or 8 and incubated for a further 2 hours before harvesting. Total RNA extracted from mycelia was used to synthesise cDNA. Primers used in the analyses are listed in Table 2.3. Absolute values were determined by interpolation from a standard curve and normalised to the expression levels of the 40S ribosomal protein S22 (RPS22) reference gene. Y-axes represent relative expression of the gene to that of RPS22. Bars represent standard error of the mean calculated from three biological replicates. N.D.; not detectable.
All of the secondary metabolite genes were minimally expressed in all strains under all pH conditions, indicating that pH alone is insufficient to turn on secondary metabolite gene expression in E. festucae (Figure 3.25). In the wild-type strain, expression of ltmE and easA was induced under acidic pH, but surprisingly, this induction was absent in the ΔpacC mutant, suggesting that PacC may be required for the induction of these genes under acidic pH conditions - a state in which PacC is thought to be inactive (Figure 3.25B and C). However, the indication that PacC is required for expression of these genes is complicated by the lack of induction of these genes in the pacCCA mutant, suggesting that over-activation of PacC may in fact prevent the upregulation of these genes (Figure 3.25B and C). The expression of easA at neutral and alkaline pH was ~4 times lower in wild-type than in the ΔpacC mutant strain, suggesting that alkaline pH- activated PacC may act to repress the easA gene. However, the pacCCA mutant did not show a greater repression of easA than wild-type, which would be expected if PacC was acting as a repressor (Figure 3.25B and C).
The expression of dmaW was upregulated in both wild-type and ΔpacC mutant under alkaline pH condition, indicating that this upregulation is alkaline pH- but not PacC- dependent (Figure 3.25D). This upregulation, however, was not seen in the pacCCA mutant. In the case of lpsA, a general trend of increasing expression with increasing pH was observed in all three strains, suggesting that the upregulation of this gene may also be alkaline pH-dependent but PacC-independent (Figure 3.25E). Lastly, the expression of perA in wild-type and pacCCA mutant remained relatively unchanged across the different pH conditions (Figure 3.25F). In the ΔpacC mutant, however, expression of perA in acidic and neutral pH was only half that of wild-type and pacCCA strains, indicating a repressive role for PacC under acidic conditions (Figure 3.25F). These results do not provide strong evidence for PacC regulation of secondary metabolite genes in E. festucae; but indicate that some genes can be alkaline pH-induced but PacC-independent, and also suggest some active role of PacC at acidic pH in E.
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festucae. However, the genes were very lowly expressed and the differences observed between treatments and strains were small; thus it is possible that the differences were due to sample variability instead of a bona fide effect of pH or mutations in pacC.
3.4.2 Expression analysis of secondary metabolite genes in planta
The secondary metabolite genes in E. festucae are naturally repressed in axenic culture, due in part to global silencing effects such as heterochromatic repression of genes. Therefore, the role of PacC in regulating the secondary metabolite genes was also investigated in planta, where heterochromatic gene silencing effects are abolished (Chujo & Scott, 2014). To this end, L. perenne plants were artificially infected with wild- type and pacC mutant strains, and the expression of secondary metabolite genes was determined in the pseudostem regions of endophyte-infected plants.
In the wild-type strain, expression of all secondary metabolite genes was considerably higher in planta than in vitro, ranging from a 60-fold difference in the expression of perA to a 1000-fold difference in the expression of easA and dmaW (Figure 3.26). Analysis of the steady-state mRNA levels in this strain showed the expression of pacC to be 0.14- fold of RPS22 in the plant, closely similar to the expression level in vitro which was 0.13-fold of RPS22 at pH 6.5 (Figure 3.27A). A measurement of the apoplast fluid pH taken from pseudostem regions of a wild-type-infected plant revealed a pH of 6.12 (Section 3.5; Table 3.2). These results indicate that the apoplast environment where E. festucae grows in has a pH that is close to neutral. The expression of pacC in the complemented mutant was relatively similar at 0.16-fold of RPS22 and some expression of pacC was detectable in the ΔpacC mutant, likely due to contamination of the plant samples (Figure 3.27A). The expression of pacCCA in the pacCCA mutant was also relatively similar in planta and in vitro, at about 0.08 and 0.10-fold the expression of RPS22, respectively (Figure 3.27A). The interpretation of these results, however, assumes that there is no difference in the expression levels of RPS22 and EF-2in planta and in vitro.
Analysis of the steady-state levels of the ltm genes (ltmE, ltmP and ltmM) showed that there was no difference in the expression of these genes among wild-type, ΔpacC, and pacCCA-infected plants (Figure 3.27B to D). Analysis of easA and dmaW also showed no difference among the strains (Figure 3.27E and F). The expression of lpsA was slightly reduced in the pacC mutants compared to wild-type, however this reduction was also
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present in the complemented strain (Figure 3.27G). Only one gene, perA, appeared to be differently expressed and downregulated in the pacCCA mutant (Figure 3.27H). Taken together with results from the in vitro expression analyses, these findings do not support the hypothesis that PacC regulates secondary metabolite pathways in E. festucae.
Figure 3.26. Expression of secondary metabolite genes in wild-type E. festucae in culture and
in planta. Differences in the expression of secondary metabolite biosynthetic genes by wild- type E. festucae in axenic culture and in planta, shown relative to the levels of (A) RPS22 and (B) EF-2 reference genes. Values were determined by interpolation from a standard curve, Y-axes represent relative mRNA abundance to the RPS22 or EF-2 reference gene transcripts. Bars represent standard error of the mean calculated from three biological replicates. N.D.; not detectable.