Simulación de las camisetas serigrafiadas en ARENA

In both experiments and under all conditions the growth rate of LU132 was consistently faster than the growth rate of LU140. This confirms earlier findings regarding the WT and acted as a positive control for the whole trial.

The fastest growth rates for all treatments and all strains were found on PDA pH 5 in complete darkness. The growth rates were reduced on the same medium with constant light and all treatments displayed their slowest growth rates on PDA pH 2.7 with constant light (Table 4.3). This also confirms earlier findings with the WT, where light exposure and low pH reduced the growth rates.

Table 4.3 Average growth rates of WT and ∆serf mutants.

Strain pH 5.0, dark pH 5.0, light pH 2.7, light Grand mean*

LU132 WT 21.77 a 21.04 a 16.39 a 19.73 a LU132 A 21.15 b 18.68 c 14.28 c 18.04 c LU132 B 21.27 b 20.20 b 15.90 b 19.12 b LU132 C 21.23 b 20.12 b 15.89 b 19.08 b LU140 WT 20.54 c 18.31 cd 13.27 de 17.37 d LU140 D 20.57 c 17.58 e 12.99 e 17.05 d LU140 E 20.62 c 17.91 de 13.39 d 17.31 d LU140 F 20.68 c 18.03 d 13.18 de 17.30 d l.s.d. 0.219 0.404 0.337 0.337†

Values are averages of two experiments with four replicates. Different letters within a column represent significantly different values (P<0.05). * _{Grand mean is the average of the three}

conditions. † _{The grant mean l.s.d. was determined using the split-plot design.}

On average, LU140 grew 12% slower than LU132. The growth rates of the LU140 ∆serf

mutants were slightly less than the growth rate of the LU140 WT (0.9% on average) but this was not statistically significant (P<0.05). The growth rates of the LU132 ∆serf mutants varied significantly from the LU132 WT (Figure 4.18). Two LU132 ∆serf mutants (B and C)

displayed a growth rate reduction of 3% while one mutant (A) had a reduction of 9%

compared to the LU132 WT. Even the slowest growing LU132 mutant (A) grew on average significantly faster than LU140 WT.

Figure 4.18 Average growth rates of mutants and WT.

l.s.d. = least significant difference of means (P<0.05). The deletion of serf in LU140 resulted in mutants (D, E and F) with similar growth rates as LU140. All LU132 ∆serf mutants (A, B and C) showed reduced growth rates. Even though, this effect was greater in mutant A, this mutant still grew significantly faster than LU140.

4.3.5.3 Phenotype microarray

The metabolic profiles and conidiation of LU132, LU140 and the six ∆serf mutants on Biolog FF plates (Biolog Inc.) were analysed by cluster analysis to group the strains. As none of the eight strains conidiated before 60 h after inoculation, the earlier time points were omitted from conidiation analysis.

As Figure 4.19 shows, the cluster analysis of the OD750 data (mycelial growth) resulted in two

main groups. One group contained LU132 WT and its ∆serf mutants B and C while the second group consisted of LU140 WT, its mutants D, E and F and LU132 mutant A. The two groups separated at a similarity distance of 0.49 (a value of 1 means complete similarity and 0 means complete dissimilarity). Analysis of the conidiation resulted in the same two groups that separated at a similarity distance of 0.54. The OD490 data (catabolic activity) was not so

clearly grouped. One strain (LU132 mutant C) separated from the others at a similarity distance of 0.59 and the remaining strains were separated into two groups at a similarity distance of 0.67. One group contained LU132, its mutant B and the two LU140 mutants E and

Figure 4.19 Cluster analysis of Phenotype microarray data.

The eight strains were clearly separated into two groups regarding their mycelial growth (OD750) and conidiation on 95 different nutrient sources. The OD490 data was more

homogeneous, resulting in higher similarity distances at the branching nodes of the dendrogram.

The experiments in this section show that the phenotypes of the six ∆serf mutants of LU132 and LU140 were not identical. All three LU140 mutants had similar phenotypes to LU140 WT. LU132 mutants B and C only showed reduced growth rates on agar plates but similar growth and conidiation characteristics as LU132 WT on Biolog plates. In addition to a reduced growth rate, LU132 mutant A also displayed a changed conidiation pattern on agar plates. These differences resulted in mutant A being grouped with LU140 WT and its mutants in the cluster analysis of the Biolog data.

4.4 Discussion

To study what impact SNP1 in the LU132 serf gene might have on its phenotype, the gene was deleted in LU132 and LU140. The hypothesis for this mutational analysis was that if the SERF function was changed by SNP1 in LU132 and this was the only molecular difference with phenotypic effect, the mutant phenotypes would be identical.

Three positively confirmed Δserf mutants were generated for each isolate. Mutants

maintained a stable antibiotic resistance and therefore a stable mutation after repeated sub- culturing. The wild types of LU132 and LU140 were confirmed to contain one copy of serf

each that was completely replaced with the knock-out cassette in the mutants and all mutants had identical sequences in the whole area subjected to DNA manipulation.

The main result of the mutant characterisation was that the mutants were not identical.

Deletion of serf did not significantly affect the LU140 mutant’s phenotypes. This implies that the LU140 variant of serf does not have a housekeeping function and does not play a

regulatory role under the conditions tested. Conversely, the LU132 variant of serf (containing SNP1) seemed to be partly responsible for the faster growth rate of LU132, as the LU132 mutants exhibited reduced growth rates. The possible function of the SERF protein was discussed in 3.4 and included involvement in photoreception, hyphal growth and cell

development. The reduced growth rates of the LU132 mutants endorsed these protein function predictions. Because the growth rates of the LU132 mutants were not completely reduced down to the level of LU140, it is likely that SNP1 in LU132 is not the only reason for the phenotypic differences between LU132 and LU140. Epigenetic differences in other parts of the genome between LU132 and LU140 would not have been changed by the mutational analysis and could therefore be maintained in the mutants and could have led to the observed results.

The evident phenotypic differences between the three LU132 mutants (A, compared to B and C) were intriguing, as all mutants were found to have identical sequences in the manipulated genomic region and all contained exactly one copy of the knock-out cassette. Mutant A of LU132 exhibited the slowest growth rate of all LU132 mutants and had also similar

conidiation characteristics as LU140. It could be possible that additional random mutations were introduced in mutant A during the transformation process. The randomness of this

Another possible explanation could be that LU132 WT naturally contained a virus or a plasmid that only got lost in A during the mutation process. This could not only explain A’s difference to B and C, but also why A’s phenotype was more similar to LU140’s phenotype. If LU132 WT harboured an extra-chromosomal element that was absent in LU140 WT, than this could be an additional cause for the phenotypic differences of the two isolates.

Because of the diverse nature of the mutant phenotypes, it was decided not to attempt gene complementation. Restoring the LU132 serf variant (with SNP1) in LU132 might have resulted in the original LU132 WT phenotype and confirm the effect of SNP1. However, it would not explain why the phenotypes of the LU132 mutants were not identical to the LU140 WT.

The results of this chapter show that a single nucleotide polymorphism alone was not the cause for the different phenotypes of LU132 and LU140. Even though it was likely to have some impact, there were other possible explanations to be explored, such as the presence of a virus or a plasmid in either isolate or epigenetic variations between the isolates.