An experimental approach to studying the role of Horizontal Gene Transfer in shaping fungal secondary metabolism

The in-silico discovery rate of putative secondary metabolite gene clusters in filamentous fungi is on a surge. With it come increasingly many reports of Horizontal Gene Transfer (HGT) of these gene clusters. Such events are inferred from discrepancies in the synteny, nucleotide composition or taxonomical
distribution of the gene clusters in question. Clustered genes have been reported to undergo HGT more often than unclustered genes, for which various hypotheses can be offered. This ’genetic sharing economy’ is very interesting from an evolutionary perspective, and yet in-silico studies leave fundamental
questions about the functional implications of HGT unanswered. Our study takes an experimental approach to studying the role of HGT in shaping fungal secondary metabolism, focusing on the immediate impact of a whole-cluster HGT event on the recipient. Four naphto-y-pyrone gene clusters - two confirmed and two putative - were selected from four different native fungal hosts (Fusarium graminearum, Fusarium fujikuroi, Trichophyton rubrum, and Aspergillus eucalypticola). Each cluster was transferred to the same locus in host organism Aspergillus nidulans using a one-step PCR-based in-vivo recombination method. Alongside the clusters, a second library was created in which the cluster pathway-specific transcription factors were overexpressed in a
second locus. In a third library, the transcription factors were overexpressed in strains lacking the cluster.
Transcriptional activity of the clusters was investigated using RT-qPCR to address splicing and transregulatory interactions as two of the potential barriers to the functional expression of HGT gene clusters in the new host. Metabolic profiling of the libraries was done by HPLC-UV/Vis-High Resolution MS to screen
for production of pathway products and intermediates, as well as other novel metabolites arising from cross-chemistry or cross-regulation.
This case study, on a small scale, demonstrates the added value of in-vivo experimental work to complement in-silico findings and hypotheses, offering additional insights into the role of HGT in fungal secondary metabolism, as well as providing an additional tool for cluster validation.