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Filamentous fungi produce various bioactive compounds that are biosynthesized by sets of proteins encoded in biosynthesis gene clusters (BGCs). For an unknown reason, many BGCs are transcriptionally silent in laboratory conditions, which has hampered the discovery of novel fungal compounds. The transcriptional reactiveness of fungal secondary metabolism is not fully understood. To gain the comprehensive view, we conducted comparative genomic and transcriptomic analyses of nine closely-related species of Aspergillus section Fumigati (A. fumigatus, A. fumigatiaffinis, A. novofumigatus, A. thermomutatus, A. viridinutans, A. pseudoviridinutans, A. lentulus, A. udagawae, and Neosartorya fischeri). For expanding our knowledge, we newly sequenced genomes of A. viridinutans and A. pseudoviridinutans, and reassembled and reannotated the previously released genomes of A. lentulus and A. udagawae. Between 34 and 84 secondary metabolite (SM) backbone genes were identified in the genomes of these nine respective species, with 8.7–51.2% being unique to the species. A total of 247 SM backbone gene types were identified in the nine fungi. Ten BGCs are shared by all nine species. Transcriptomic analysis using A. fumigatus, A. lentulus, A. udagawae, A. viridinutans, and N. fischeri was conducted to compare expression levels of all SM backbone genes in four different culture conditions; 32–83% of SM backbone genes in these species were not expressed in the tested conditions, which reconfirmed that large part of fungal SM genes are hard to be expressed. The species-unique SM genes of the five species were expressed with lower frequency (18.8% in total) than the SM genes that are conserved in all five species (56%). These results suggest that the expression tendency of BGCs is correlated with their interspecies distribution pattern. Our findings increase understanding of the evolutionary processes associated with the regulation of fungal secondary metabolism.

The filamentous fungal pathogen Aspergillus fumigatus is one of the most common causal agents of invasive fungal infection in humans; the infection is associated with an alarmingly high mortality rate. In this study, we investigated whether a mycovirus, named AfuPmV-1M, can reduce the virulence of A. fumigatus in a mouse infection model. AfuPmV-1M has high sequence similarity to AfuPmV-1, one of the polymycovirus that is a capsidless four-segment double-stranded RNA (dsRNA) virus, previously isolated from the genome reference strain of A. fumigatus, Af293. However, we found the isolate had an additional fifth dsRNA segment, referred to as open reading frame 5 (ORF5), which has not been reported in AfuPmV-1. We then established isogenic lines of virus-infected and virus-free A. fumigatus strains. Mycovirus infection had apparent influences on fungal phenotypes, with the virus-infected strain producing a reduced mycelial mass and reduced conidial number in comparison with these features of the virus-free strain. Also, resting conidia of the infected strain showed reduced adherence to pulmonary epithelial cells and reduced tolerance to macrophage phagocytosis. In an immunosuppressed mouse infection model, the virus-infected strain showed reduced mortality in comparison with mortality due to the virus-free strain. RNA sequencing and high-performance liquid chromatography (HPLC) analysis showed that the virus suppressed the expression of genes for gliotoxin synthesis and its production at the mycelial stage. Conversely, the virus enhanced gene expression and biosynthesis of fumagillin. Viral RNA expression was enhanced during conidial maturation, conidial germination, and the mycelial stage. We presume that the RNA or translation products of the virus affected fungal phenotypes, including spore formation and toxin synthesis. To identify the mycovirus genes responsible for attenuation of fungal virulence, each viral ORF was ectopically expressed in the virus-free KU strain. We found that the expression of ORF2 and ORF5 reduced fungal virulence in the mouse model. In addition, ORF3 affected the stress tolerance of host A. fumigatus in culture. We hypothesize that the respective viral genes work cooperatively to suppress the pathogenicity of the fungal host.

Fungi are a rich source of natural products with biological activities. In this study, we evaluated viral effects on secondary metabolism of the rice blast fungus Magnaporthe oryzae using an isolate of APU10-199A co-infected with three types of mycoviruses: a totivirus, a chrysovirus, and a partitivirus. Comparison of the secondary metabolite profile of APU10-199A with that of the strain lacking the totivirus and chrysovirus showed that a mycotoxin tenuazonic (TeA) acid was produced in a manner dependent on the mycoviruses. Virus reinfection experiments verified that TeA production was dependent on the totivirus. Quantitative reverse transcription PCR and RNA-sequencing analysis indicated the regulatory mechanism underlying viral induction of TeA: the totivirus activates the TeA synthetase gene TAS1 by upregulating the transcription of the gene encoding a Zn(II)2-Cys6-type transcription factor, TAS2. To our knowledge, this is the first report that confirmed mycovirus-associated regulation of secondary metabolism at a transcriptional level by viral reinfection. Because only treatment with dimethyl sulfoxide has been reported to trigger TeA production in this fungus without gene manipulation, our finding highlights the potential of mycoviruses as an epigenomic regulator of fungal secondary metabolism.