Effects of secondary metabolism on sexual development andwood decay in agaricomycetes
Takehito NAKAZAWA
Graduate School of Agriculture, Kyoto University

Research aims
Agaricomycetes are characterized by two main criteria. One is dramatic
morphological differentiation, fruiting development, or mushroom formation. Mushrooms
are relatively large multicellular structures that agaricomycetes form during sexualreproduction. The second is wood decay, especially white rot. White-rot fungi play animportant role in the global carbon cycle since they biodegrade almost all wood lignin foundin nature. Molecular genetic studies on the regulation of morphogenesis and secondary
metabolism in ascomycetes, and physiological and biochemical studies on wood decay,indicate a correlation between mushroom formation, wood decay, and the secondary
metabolism found in white-rot fungi. In this study, we investigated this hypothesis throughan extensive molecular genetics approach using the white-rot fungus Pleurotus ostreatus.
Methods
In this study, P. ostreatus strains 20b and 20b/1 (a ku80 disruptant from wild-typestrain PC9) was used for gene targeting experiments. Examination of the effects of genedisruption on fruiting development and the ability of white rot was carried out according to
the procedure described in the previous study1). Quantification of wood lignin-containing
sawdust media was performed according to the Klason method. P. ostreatus strains weregrown on sawdust media for 13 days, after which total RNA was extracted from mycelial
cells of each to perform comparative RNAseq analyses.
Results
Effects of the targeted disruption of veA and laeA genes on the ligninolytic activity in P.
ostreatus
We disrupted P. ostreatus genes homologous to ascomycete genes veA and laeA,
which were reported to affect secondary metabolism and morphogenesis in various
ascomycetes. LaeA and veA disruptions did not cause significant defects in either sexualdevelopment or ligninolytic activity (Table 1), although extracellular manganese peroxidase(MnP) activity was diminished in the veA disruptant when it was grown on GP(glucose-peptone) liquid medium.
Effects of targeted disruption of genes involved in sexual development on theligninolytic activity of P. ostreatus
We disrupted P. ostreatus genes homologous to agaricomycete Coprinopsis cinerea
genes snf5, ubc2, and rmt1 (Table 1), which were reported to be essential for sexualdevelopment. The results showed that ubc2 is essential for dikaryosis in P. ostreatus as well
as C. cinerea, while snf5 does not affect dikaryosis but is essential for fruiting developmentin P. ostreatus. Unlike the case of C. cinerea, rmt1 disruption did not cause any defects ineither fruiting initiation or hyphal growth rate in response to carbon sources in P. ostreatus.
Targeted disruption of ubc2 caused defects in wood lignin biodegradation, while disruptionof rmt1 did not. We could not examine the effects of snf5 disruption on the ability tobiodegrade wood lignin because the hyphal growth rate of snf5 disruptants was much lower
than that of the wild-type strain.
Effects of mutations that cause defects in ligninolytic activity on sexual development inP. ostreatus
We also examined the effect of disrupting genes that impede wood ligninbiodegradation on fruiting development in P. ostreatus. Pex1 and chd1 disruption, whichcause significant defects in ligninolytic activity, were also shown to be essential for fruiting
initiation, unlike wtr1 disruption, which was shown to cause slight defects in the ligninolytic
activity but no effect on fruiting initiation.
Recently, we identified some new genes likely responsible for mutant phenotype
defects in wood lignin biodegradation using the methodology demonstrated in the previous
study2). Specifically, two genes encoding putative transcription factors were identified. One
is involved in the regulation of secondary metabolism and morphogenesis in Aspergilli.
Another two genes were identified that encode putative proteins involved in nucleosome
assembly, which is one of the epigenetic regulatory mechanisms involved in the regulation of
secondary metabolisms in ascomycetes. We are now confirming that these genes are indeed
responsible for the mutant phenotypes. Furthermore, a dominant mutation that causes defects
in both lignin biodegradation and fruiting initiation was also identified, along with the gene
responsible for this mutation.
Gene lists that are significantly inactivated by snf5 and ubc2 disruption overlap withthose inactivated by pex1, chd1, and wtr1 disruptions

We investigated the effects of the gene disruptions performed in this study on gene
expression via RNAseq. Many of the genes that were significantly inactivated by snf5 andubc2 disruption, which caused defects in sexual development, were also inactivated by wtr1,chd1, and pex1.
Conclusion
This research shows that some gene disruptions that cause defects in wood ligninbiodegradation also cause defects in fruiting development. Comparative RNAseq analyses
also suggest a correlation between the regulation mechanisms of sexual development and theligninolytic system at transcription level, although it is unclear whether this is a direct effector not. Reverse genetics data acquired in this study did not support the hypothesis that themalfunction of regulation mechanisms in P. ostreatus homologous to the regulation of
secondary metabolism in ascomycetes affect sexual development and/or ligninolytic activity
in P. ostreatus. However, the extensive forward genetics study identified four genes (fourmutants) that are likely responsible for the mutant phenotype and defects in ligninolyticactivity. These genes encode putative transcription factors homologous to those involved insecondary metabolism and morphogenesis in Aspergilli. The putative proteins are involvedin nucleosome positioning, which may be involved in epigenetic regulation. Based on theseresults, we must investigate whether the regulatory mechanisms of secondary metabolism
investigated in ascomycetes are present in agaricomycetes. and the molecular mechanismsthat affect both lignin biodegradation and fruiting development.
References
1) Shioya, T. et al. (2013) The Coprinopsis cinerea septin Cc.Cdc3 is involved in stipe cellelongation. Fungal Genet. Biol. 58-59: 80–90.
2) Nakazawa, T. et al. (2017) Identification of two mutations that cause defects in theligninolytic system through an efficient forward genetics in the white-rot agaricomycete
Pleurotus ostreatus. Environ. Microbiol. 19: 261–272.

Table 1. Effects of gene disruptions on the ligninolytic activity and sexual development of P.
ostreatus.
Disrupted gene
Effect on the ligninolytic activity
Effect on sexual development
pex1
significant decrease
defects in fruiting initiation
wtr1
slight decreaseno effect
chd1
significant decrease
defects in fruiting initiationveA
no effectno effectlaeA
no effectno effect1
snf5
no effect
defects in fruiting initiation
ubc2
decrease
defects in dikaryosis
rmt1
no effectno effectpcc1
no effect
defects in dikaryosis
1
Effects of snf5 disruption on gene expression pattern are very similar those of pex1, wtr1
and chd1.