Overexpressing Pleurotus ostreatus rho1b results in transcriptional upregulation of the putative cellulolytic enzyme‐encoding genes observed in ccl1 disruptants

Abstract

Summary The transcriptional expression pattern of lignocellulolytic enzyme‐encoding genes in white‐rot fungi differs depending on the culture conditions. Recently, it was shown that 13 putative cellulolytic enzyme‐encoding genes were significantly upregulated in most Pleurotus ostreatus ligninolysis‐deficient mutant strains on beech wood sawdust medium. However, the mechanisms by which this transcriptional shift is triggered remain unknown. In this study, we identified one mechanism. Our previous study implied that histone H3 N‐dimethylation at lysine 4 level possibly affects the shift; therefore, we analysed the expression pattern in the disruptants of P. ostreatus ccl1, which encodes a putative component of the COMPASS complex mediating the methylation. The results showed upregulation of 5 of the 13 cellulolytic enzyme‐encoding genes. We also found that rho1b, encoding a putative GTPase regulating signal transduction pathways, was upregulated in the ccl1 disruptants and ligninolysis‐deficient strains. Upregulation of at least three of the five cellulolytic enzyme‐encoding genes was observed in rho1b‐overexpressing strains but not in ccl1/rho1b double‐gene disruptants, during the 20‐day culture period. These results suggest that Rho1b may be involved in the upregulation of cellulolytic enzyme‐encoding genes observed in the ccl1 disruptants. Furthermore, we suggest that Mpk1b, a putative Agaricomycetes‐specific mitogen‐activated protein kinase, functions downstream of Rho1b.

Pleurotus ostreatus as a model mushroom in genetics, cell biology, and material sciences

“…Based on these findings, we isolated single-gene deletions related to histone modifications. Altered expression patterns were observed for some gene deletions (Okuda et al 2021 , unpublished). Future studies are required to investigate how each regulator alters transcriptional expression, which will provide a methodology for the artificial induction and regulation of ligninolytic, cellulolytic, and hemicellulolytic systems.…”

“…However, even though efficient gene-targeting is available for P. ostreatus today, it is still laborious and time-consuming to analyze the precise effects of the deletion/disruption of a certain gene on fruiting development. This is because dikaryotic strains, in which a target gene is deleted/disrupted in both nuclei, should be generated (Nakazawa et al 2017b ; Okuda et al 2021 ). Recently, gene targeting in both nuclei of a dikaryon in a single transformation procedure with plasmid-based CRISPR/Cas9 was reported (Yamasaki et al 2022 ), which would allow for quick and efficient molecular breeding.…”

A Molecular Genetics Approach to Elucidating the Mechanisms Underlying Lignin Degradation by White-Rot Fungi

“…The effects of defects in peroxisome biogenesis (pex1 deletion) on lignin-degrading abilities and the utilization of carbon sources have also been analyzed 34) . Recently, the effects of histone modification on the transcriptional expression of wood-degrading enzyme-encoding genes were analyzed using ccl1 deletants 35) . However, this methodology may not be applicable to various other white-rot fungi, as it requires the generation of ku or lig4 deletant from the wild-type strain by homologous recombination.…”

Systems biology-guided understanding of white-rot fungi for biotechnological applications: A review

“…Identifying transcription factors in WRF is essential to understand and control gene regulation for further genetic engineering efforts. Though the development of efficient genetic tools in WRF is still in its infancy, a series of knockout strains from Pleurotus ostreatus have been recently built to probe the role of putative transcription factors in lignin degradation more precisely ( Okuda et al., 2021 ; Wu et al., 2020 ). Specifically, two putative transcription factors, wrt1 and gat- 1, and one chromatin remodeler, chd1, were knocked out or mutated ( Wu et al., 2020 ).…”

“…Co-expression data revealed another zinc-finger transcription factor that might also play a role in regulation. A follow-up study implies a different mechanism involving a putative mitogen-activated protein kinase (MAPK) cascade ( Okuda et al., 2021 ). Disruption of the MAPK cascade – whether through deletion of a gene encoding for a protein kinase, rho1b , or its downstream acting kinase, mpk1 – resulted in disrupted upregulation of several CAZyme-encoding genes in the AA9 or GH7 families compared to parental strains grown in the same conditions or compared to a rho1b- overexpressing strain.…”