Farnesol-induced hyperbranched morphology with short hyphae and bulbous tips of Coriolus versicolor

Abstract

As the first fungal quorum sensing molecule, farnesol-induced morphological transition is usually studied in dimorphic fungi, but in basidiomycetes the morphological changes regulated by farnesol are rarely investigated. In this study, we found that farnesol made the basidiomycete Coriolus versicolor develop into a hyperbranched morphology with short hyphae and bulbous tips. Farnesol treatment resulted in a significant increase of intracellular oxidative stress level, which influenced the expression of several morphogenesis-related genes, and thereby led to the morphological changes. High oxidative stress level significantly stimulated the expression of laccase genes for improving intracellular laccase biosynthesis. The resulted hyperbranched morphology further accelerated the secretion of intracellular laccase into culture medium. As a result, extracellular laccase production reached a maximum of 2189.2 ± 54.7 U/L in farnesol-induced cultures, which was 6.8-fold greater than that of control cultures. SDS-PAGE and native-PAGE showed that farnesol increased laccase production by promoting the biosynthesis of three laccase isoforms. Together these results provide new opportunities in not only understanding the farnesol-regulated mycelial morphology in basidiomycetes, but also developing novel strategies for enhancing the production of secreted enzymes of biotechnological interest.

Figure 1

Morphological changes of C. versicolor at 96 h after inoculation in the presence and absence of farnesol (final concentration, 4.0 mM) in submerged cultures. Column A: macroscopic phenotype; Column B: microscopic phenotype with 10× magnification; Column C: microscopic phenotype with 100× magnification. Bar, 100 um; Column D: microscopic phenotype with 200× magnification. Bar, 50 um; Column E: microscopic phenotype with 1,000× magnification. Bar, 10 um; Column F: SEM tests with 3,000× magnification; Column G: SEM tests with 20,000× magnification.

Figure 2

The growth of mycelium pellet treated (or not) with farnesol on culture dish with PDA.

Figure 3

Effect of farnesol on cell growth during C. versicolor submerged cultures. The results are average of 3 replicate experiments. Error bars correspond to standard deviation.

Figure 4

The relative expression levels of morphogenesis-related genes induced by farnesol during C. versicolor submerged cultures. (a) rhoA, hog1 and tup1; (b) racA and cftA. The transcription levels of genes in farnesol-induced cultures were relative to that in control cultures without farnesol addition. The values represent the number of times each gene is expressed in induction group compared to control group (set at 1.0). Values are means of 3 replicates. Error bars correspond to standard deviation.

Figure 5

Time course of intracellular ROS and GSSG relative levels induced by farnesol during C. versicolor submerged cultures. The induction levels of ROS and GSSG in farnesol-induced cultures were relative to that in control cultures without farnesol addition. The values represent the number of times ROS and GSSG were induced compared to control group (set at 1.0). Values are means of 3 replicates. Error bars correspond to standard deviation.

Figure 6

(a) Effect of farnesol on intracellular laccase activity and extracellular laccase activity during C. versicolor submerged cultures. Symbols: □, intracellular laccase activity (control); ■, intracellular laccase activity (farnesol); ○, extracellular laccase activity (control); ●, extracellular laccase activity (farnesol). (b) Effect of farnesol on extracellular protein concentration and extracellular laccase specific activity during C. versicolor submerged cultures. Symbols: Δ, extracellular protein concentration (control); ▲, extracellular protein concentration (farnesol); ∇, extracellular laccase specific activity (control); ▼, extracellular laccase specific activity (farnesol). The results are average of 3 replicate experiments. Error bars correspond to standard deviation.

Figure 7

(a) SDS-PAGE pattern of extracellular culture supernatants (20 uL) with silver staining. Electrophoresis was carried out using a 12% cross-linked polyacrylamide gel. Lane 1: cell-free supernatants in control cultures; Lane 2: cell-free supernatants in farnesol-induced cultures; Lane 3: laccase standard from C. versicolor (Sigma); Lane 4: molecular mass marker proteins. (b) Native-PAGE pattern of extracellular culture supernatants (20 uL) with activity staining. After native-PAGE the laccase isozymes were stained with catechol. Lane 1: cell-free supernatants in farnesol-induced cultures; Lane 2: cell-free supernatants in control cultures.

Figure 8

Time course of the relative expression levels of laccase genes (lcc1, lcc2 and lcc3) induced by farnesol. The transcription levels of genes in farnesol-induced cultures were relative to that in control cultures without farnesol addition. The values represent the number of times each gene is expressed in induction group compared to control group (set at 1.0). Values are means of 3 replicates. Error bars correspond to standard deviation.