Hyphal heterogeneity in Aspergillus niger

Mycelial fungi use hyphae to colonize substrates. These hyphae secrete enzymes that convert complex polymers into breakdown products that can be taken up to serve as nutrients. Using GFP as a reporter it has been shown that exploring hyphae of Aspergillus niger are heterogenic with respect to expression of the glucoamylase gene glaA; some hyphae strongly express the glucoamylase gene glaA, while others express it lowly. This was a surprising finding considering the fact that all hyphae were exposed to similar environmental conditions. Apparently, a vegetative mycelium is more complex than generally assumed. In this thesis it is shown that the expression of other genes encoding secreted enzymes is also heterogenic in A. niger. Co-expression studies, using GFP and dTomato as reporters, showed that hyphae that highly express one of these genes also highly express other genes encoding secreted proteins. Over and above this, high expression of genes encoding secreted proteins correlated with high expression of a gene involved in central metabolism and with high ribosomal RNA content. This suggests that there are two populations of hyphae at the periphery that differ in their transcriptional and translational activities. These studies were extended with whole genome transcription profiling of individual hyphae. In fact, this was the first single cell analysis in a microbe. In order to perform (sub)- cellular transcriptomics on exploring hyphae, protocols have been set up to collect individual hyphae using LPC, to isolate RNA and to amplify cDNA. Microarray analysis led to the conclusion that exploring hyphal tips have a different RNA content compared to the rest of the periphery. Furthermore, neighboring hyphal tips were shown to be highly heterogenic in their expression of genes encoding for all kinds of functions. This suggests that heterogeneity is caused by stochastic gene expression and / or by epigenetic processes. A role of epigenetic processes was also indicated by the changes in gene expression that were observed upon treatment with the chemicals AZC and butyrate. Finally, it is shown that heterogeneity in the colony is not the result of heterogeneity in the inoculum but develops during vegetative growth. It is also demonstrated, using the novel COPAS and LMPC techniques, that heterogeneity is not restricted to cm-scale colonies but can also be found within and between sub-millimeter micro-colonies grown in liquid shaken culture