In this project we have studied six genes with a putative role in trehalose synthesis in A. niger: tpsA, tpsB, tpsC, tppA, tppB and tppC. All six genes encode homologous proteins and no similar gene products within the A. niger genome could be detected. Three proteins, TpsA, TpsB and TpsC, have previously been identified as orthologs to the yeast protein Tps1. As the orthologs are conserved in related species, it is plausible that there is a functional differentiation between the paralogs, e.g. one paralog could be essential for trehalose synthesis in conidia, whereas another paralog is strictly induced by stress. This assumption is in line with the previous observation in A. niger where the expression of tpsB is stress-induced whereas tpsA is constitutively expressed , although our data also suggest that tpsB has a role during differentiation (see Figure 3). When deleting the trehalose-phosphate-synthase paralogs, only ΔtpsA displayed a reduced trehalose content. The lower level in this mutant is in line with a previous report using a different target strain and deletion procedure . In the related fungus, A. fumigatus, a tpsA/tpsB double deletion resulted in a strain with depleted trehalose content, and in the same study, it was shown that the expressions of tpsC and –D were very low at all time points . These authors evaluated their expression data using RNA extracted from hyphae, and in the present study, the A. niger tpsC was expressed at very low levels at 72 h. Thus the results from the two fungi are not contradictory, and most likely an A. niger tpsA/tpsB deletion mutant would also have a depleted trehalose content. The results from A. niger and A. fumigatus are also in accordance with findings in A. nidulans where deletion of tpsA resulted in depleted trehalose content , as that species does not have the tpsB paralogue. A conclusion from studying the trehalose content from these three species is that TpsA is the most important trehalose-phosphate-synthase under normal conditions, but lack of the tpsA gene can be fully compensated by TpsB in A. fumigatus and partly by at least one of TpsB or TpsC in A. niger, but not by TpsD in A. nidulans.
The deletion mutant with the most distinctive characteristics in our experiments was ΔtppA, i.e. with an abnormal morphology and reduced levels of both trehalose-6-phosphate and trehalose. The altered morphology of the strain is probably due to toxicity of T6P as indicated for the corresponding deletion mutant in A. fumigatus. However, in A. niger as well as A. fumigatus and A. nidulans[12, 25], mutants of tppA are not totally lacking in trehalose. Therefore, it is possible that under specific conditions, e.g. when TppA is absent, TppB, and also TppC where present, may contribute to some T6P activity. Another possibility is that the sugar can be synthesized by proteins other than Tps/Tpp, e.g. the Trehalose Phosphorylase pathway, for which putative genes have been identified and partially characterized in N. crassa and A. fumigatus and also exist in A. niger (ANI_1_2720024). However, it is possible to generate mutants, within the homologous Tps/Tpp group, in A. fumigatus and A. nidulans that totally lack trehalose [11, 12]. Therefore, we believe that this is the only active trehalose synthesis pathway in Aspergilli. However, internal trehalose contents may not solely be dependent on the presence and expression of these six genes, as in S. cerevisiae there is a strong linkage between trehalose synthesis and the degrading trehalases  as well as evidences of posttranscriptional activation of the genes involved in trehalose metabolism [42, 43].
Besides a putative phosphatase activity, TppB and TppC may have similar biological roles as the yeast proteins Tps3 and Tsl1, which also contain phosphatase domains – in yeasts, deletion of both genes is necessary before some reduction in internal trehalose content can be observed . It is intriguing that tpsB and tppC are linked on the chromosome. We cannot explain why the conidial trehalose content in this double mutant was significantly higher after 28 days, but based on the expression patterns (see Figure 3), it is possible that the expression of the two genes are regulated by the same factors. In addition to the above-mentioned observations, some conclusions can be drawn from the gene expression data: All identified genes were expressed, indicating that the paralogs are not inactive duplicates. For tpsC and tppB, the expressions were consistently low after 6 h, indicating that the two genes may be regulated by the same mechanism. This assumption is supported by a previous observation using A. oryzae arrays where the tpsC and tppB orthologs were down-regulated in a deletion strain of atfA, a gene encoding a transcription factor . To our knowledge, two previous studies describing the expression of trehalose synthesis genes in A. niger during germination, using microarray technology, or in combination with RNA sequencing, have been published [29, 45]. With the exception that van Leeuwen and co-workers  saw a drastic drop after 2 h and then a gradual up-regulation of tpsA and tpsB, those results are in line with our findings.
The extensive measurements of internal trehalose indicate that the trehalose contents, for all strains, were low in 5 day old conidia, significantly elevated in 14 day old conidia, and then maintained at the value of 14 days (Figure 7). A plausible hypothesis is that conidia of A. niger reach full maturity, at least in terms of trehalose accumulation, sometime between 5 days and 2 weeks. Consequently it is not advisable to perform stress experiments on young conidia because their trehalose content is not necessarily typical for the final level, especially not in kusA deficient strains that seem to have slower conidial maturation in terms of trehalose content.
We found that 2 week old conidia of ΔtppB were more susceptible to heat shock than wild-type conidia, indicating that trehalose protects the spores from thermal stress. These results are in line with earlier studies in Aspergillus species [11, 12, 23]. However, in contrast to results from A. fumigatus and A. nidulans, we could not detect any increased sensitivity of ΔtppB to oxidative stress [11, 12], salt or acid stress, or any decreased viability after long term storage. It should be noted that unlike ΔtppB in our experiments, which harbored approximately one third of wild-type trehalose content, the A. fumigatus and A. nidulans mutants were totally depleted of trehalose.
In S. cerevisiae it has been shown that, using a two-hybrid assay, the four homologous proteins physically interact. When repeating the experiments using the six identified A. niger proteins, we could observe interactions for four of six proteins. These results suggest that TppA and TpsA-C form a complex, while the phylogenetically more distant proteins, TppB and TppC, are present outside the complex. However, due to the experimental limits, it is possible that neither TppB nor TppC was correctly folded and therefore not interacting. It is notable that in S. cerevisiae, a truncated version of Tsl1 was necessary for the success of the interaction experiments , in contrast to our experiment in which we only used full-length proteins.