No abstract

Stipe elongation is an important process in the development of the fruiting body and is associated with the commodity quality of agaric fungi. In this study, F. filiformis was used as a model agaric fungus to reveal the function of the chromatin modifier gene containing the JmjC domain in stipe elongation. First, we identified a JmjC domain family gene (FfJmhy) with a 3684 bp length open reading frame (ORF) in F. filiformis. FfJmhy was predicted to have a histone H3K9 demethylation function, and was specifically upregulated during stipe rapid elongation. Further investigation revealed that the silencing of FfJmhy inhibited the mycelial growth, while overexpression of this gene had no effect on the mycelial growth. Comparative analysis revealed that the stipe elongation rate in FfJmhy overexpression strains was significantly increased, while it was largely reduced when FfJmhy was silenced. Taken together, these results suggest that FfJmhy positively regulates the mycelial growth and controls the elongation speed and the length of the stipe. Moreover, cell wall-related enzymes genes, including three exo-β-1,3-glucanases, one β-1,6-glucan synthase, four chitinases, and two expansin proteins, were found to be regulated by FfJmhy. Based on the putative functions of FfJmhy, we propose that this gene enhances the transcription of cell wall-related enzymes genes by demethylating histone H3K9 sites to regulate remodeling of the cell wall in rapid stipe elongation. This study provides new insight into the mechanism of rapid stipe elongation, and it is important to regulate the commodity quality of agaric fungi.

In real-time quantitative PCR (RT-qPCR), internal control genes (ICGs) are crucial for normalization. This study screened 6 novel ICGs: Pre-mRNA-splicing factor cwc15 (Cwf15); ER associated DnaJ chaperone (DnaJ); E3 ubiquitin-protein ligase NEDD4 (HUL4); ATP-binding cassette, subfamily B (MDR/TAP), member 1 (VAMP); Exosome complex exonuclease DIS3/RRP44 (RNB); V-type H+-transporting ATPase sub-unit A (V-ATP) from the 22-transcriptome data of 8 filamentous fungi. The six novel ICGs are all involved in the basic biological process of cells and share the different transcription levels from high to low. In order to further verify the stability of ICGs candidates, the six novel ICGs as well as three traditional housekeeping genes: β-actin (ACTB); β-tubulin (β-TUB); glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and the previously screened reference genes: SPRY-domain-containing protein (SPRYp); Ras-2 protein (Ras); Vacuolar protein sorting protein 26 (Vps26) were evaluated by geNorm and NormFinder statistical algorithms. RT-qPCR of 12 ICGs were performed at different developmental stages in Flammulina filiformis and under different treatment conditions in Neurospora crassa. The consistent results of the two algorithms suggested that the novel genes, RNB, V-ATP, and VAMP, showed the highest stability in F. filiformis and N. crassa. RNB, V-ATP, and VAMP have high expression stability and universal applicability and therefore have great potential as ICGs for standardized calculation in filamentous fungi. The results also provide a novel guidance for the screening stable reference genes in RT-qPCR and a wide application in gene expression analysis of filamentous fungi.

Flammulina filiformis is a popular edible mushroom that easily suffers from heat and oxidative stresses. The cyclic adenylate-dependent protein kinase A (cAMP/PKA) pathway is the main signaling pathway in response to environmental stress, and the PKAC is the terminal catalytic subunit of this pathway. In this study, the Pkac gene was identified in F. filiformis, which was highly conserved in basidiomycetes and ascomycetes. The transcription analysis showed that the Pkac gene was involved in the mycelial growth and the fruiting body development of fungi. In Neurospora crassa, the Pkac gene deletion (ΔPkac) resulted in the slower growth of the mycelia. We complemented the F. filiformis FfPkac to N. crassa ΔPkac mutant to obtain the CPkac strain. The mycelial growth in the CPkac strain was restored to the same level as the WT strain. In addition, the FfPkac gene showed significantly up-regulated expression under heat and oxidative stresses. By analyzing the differentially expressed genes of ΔPkac and Cpkac with WT, respectively, seven downstream genes regulated by Pkac were identified and may be related to mycelial growth. They were mainly focused on microbial metabolism in diverse environments, mitochondrial biogenesis, protein translation and nucleocytoplasmic transport. RT-qPCR results confirmed that the expression patterns of these seven genes were consistent with FfPkac under heat and oxidative stresses. The results revealed the conserved functions of PKAC in filamentous fungi and its regulatory mechanism in response to heat and oxidative stresses.