Role of the blue light receptor gene Icwc-1 in mycelium growth and fruiting body formation of Isaria cicadae

Abstract

The Isaria cicadae, is well known highly prized medicinal mushroom with great demand in food and pharmaceutical industry. Due to its economic value and therapeutic uses, natural sources of wild I. cicadae are over-exploited and reducing continuously. Therefore, commercial cultivation in controlled environment is an utmost requirement to fulfill the consumer's demand. Due to the lack of knowledge on fruiting body (synnemata) development and regulation, commercial cultivation is currently in a difficult situation. In the growth cycle of macrofungi, such as mushrooms, light is the main factor affecting growth and development, but so far, specific effects of light on the growth and development of I. cicadae is unknown. In this study, we identified a blue light receptor white-collar-1 (Icwc-1) gene homologue with well-defined functions in morphological development in I. cicadae based on gene knockout technology and transcriptomic analysis. It was found that the Icwc-1 gene significantly affected hyphal growth and fruiting body development. This study confirms that Icwc-1 acts as an upstream regulatory gene that regulates genes associated with fruiting body formation, pigment-forming genes, and related genes for enzyme synthesis. Transcriptome data analysis also found that Icwc-1 affects many important metabolic pathways of I. cicadae, i.e., amino acid metabolism and fatty acid metabolism. The above findings will not only provide a comprehensive understanding about the molecular mechanism of light regulation in I. cicadae, but also provide new insights for future breeding program and improving this functional food production.

Keywords: Isaria cicadae; blue light receptor; developmental regulation; fruiting body formation; mycelium growth.

Figure 1

The domain organization of IcWC-1 protein and the Phylogenetic tree of IcWC-1. (A) The regions corresponding to LOV, PAS domains, the nuclear localization signal (NLS) and ZnF are shown. (B) Phylogenetic tree of WC-1 homologous proteins from fungi. The amino acid sequences of WC-1 homologous proteins from different species were downloaded from the NCBI database for phylogenetic analyses (protein sequences in Supplementary Material). The topology of this tree was generated using neighbour-joining (NJ) method of MEGA7 with 1,000 bootstraps replicates. These numbers represent the percentage of replication trees (1,000 replications) with related taxa clustered together in the boot test.

Figure 2

Generation and identification Icwc-1 mutants of I. cicadae. (A) Schematic diagram of knockout ∆Icwc-1 via homologous recombination. (B) PCR validation of knockout strains. M: Maker, 1: knockout vector PCAMBIA1300-Icwc-1, 2: WT strain, 3–5: mutants. 3: ∆Icwc-1-2, 4: mutant ∆Icwc-1-8, 5: mutant ∆Icwc-1-40. (C) Schematic diagram of the generation of the complemented vector. H3 promoter in pKD5-GFP derived from Pyricularia oryzae. (D) PCR verification of complementary strains. M: Maker, 1: wild type strain WT, 2: mutant strain ∆Icwc-1-2, 3: mutant strain ∆Icwc-1-8, 4: mutant strain ∆Icwc-1-40, 5: the complementary strain ∆Icwc-1-C. (E) Expression of Icwc-1 gene in wild-type strain, mutant strain, and the complementary strain. (F) Location of Icwc-1 gene expression analysis. GFP observation under fluorescence microscope. The hyphae and spores of the wild-type strain and the ∆Icwc-1-C strain were stained with DAPI and observed under a fluorescence microscope. Bar = 10 μm.

Figure 3

Effect of Icwc-1 gene on growth characters in I. cicadae. (A) Colony characters of different strains (wild-type, ∆Icwc-1 and ∆Icwc-1-C) of I. cicadae were photographed and recorded after 10 days of alternating light and dark culture on PDA medium; (B) 5 μl of the spore suspension is inoculated on PDA medium, cultured under white light conditions for 10 days, and the colony diameter was recorded; (C) The strains were cultured on a PDA plate for 10 days at 25°C, spores were washed with water, diluted to a certain concentration and counted under a microscope; (D) Effects of Icwc-1 gene deletion on mycelial biomass of I. cicadae. The symbol “*” indicates a significant difference (p < 0.05) compared with wild-type strain. The symbol ‘**’ indicates significant difference (p < 0.01) compared with wild-type strain.

Figure 4

Effect of Icwc-1 gene deletion on expression of genes related to mycelial metabolism and fruiting body formation of I. cicadae. (A) The relative expression of genes of wild-type strain and Icwc-1 mutant strain in I. cicadae detected by RT-PCR. (B) Fruiting body formation of I. cicadae (WT, ∆Icwc-1 and ∆Icwc-1-C). The symbol “*” indicates a significant difference (p < 0.05) compared with the original strain, while “****” indicates an extremely significant difference (p < 0.01). Gene expression profile regulated by Icwc-1 gene.

Figure 5

Comparative gene ontology (GO) analysis of the differentially expressed genes (DEGs) between the transcriptome of wild type and Icwc-1 mutants. (A) The volcano plot is two-dimensional, with the y-axis representing the negative log10 of the adjusted value of p, and the x-axis displays the variation ratio of each gene (log2 FC). The green spots represent significantly down-regulated genes in the mutants, while the red spots indicate up-regulated genes [false discovery rate (FDR) < 0.05]. Gray spots represent the genes that did not show differential expression. (B) The heat map shows the gene expression patterns in Icwc-1 mutants and wild-type (WT) of I. cicadae. (C) The results are summarized in three main GO categories (Cellular Component, Molecular Function and Biological Process). The x-axis indicates different mutants. The y-axis indicates the GO term. (D) KEGG enrichment analysis of the DEGs between the transcriptome of I. cicadae wild-type and Icwc-1 mutants. The y-axis indicates the KEGG pathway. The x-axis indicates the gene ratio.