MAC Family Transcription Factors Enhance the Tolerance of Mycelia to Heat Stress and Promote the Primordial Formation Rate of Pleurotus ostreatus

Abstract

Pleurotus ostreatus is a typical tetrapolar heterologous edible mushroom, and its growth and development regulatory mechanism has become a research hotspot in recent years. The MAC1 protein is a transcription factor that perceives copper and can regulate the expression of multiple genes, thereby affecting the growth and development of organisms. However, its function in edible mushrooms is still unknown. In this study, two transcription factor genes, PoMCA1a and PoMAC1b, were identified. Afterwards, PoMAC1 overexpression (OE) and RNA interference (RNAi) strains were constructed to further explore gene function. The results showed that the PoMAC1 mutation had no significant effect on the growth rate of mycelia. Further research has shown that OE-PoMAC1a strains and RNAi-PoMAC1b strains exhibit strong tolerance under 32 °C heat stress. However, under 40 °C heat stress, the OE of PoMAC1a and PoMAC1b promoted the recovery of mycelial growth after heat stress. Second, the OE of PoMAC1a can promote the rapid formation of primordia and shorten the cultivation cycle. In summary, this study indicated that there are functional differences between PoMAC1a and PoMAC1b under different heat stresses during the vegetative growth stage, and PoMAC1a has a positive regulatory effect on the formation of primordia during the reproductive growth stage.

Keywords: Oyster mushroom; fruiting body; gene overexpression; gene silencing; stress tolerance.

Figure 1

Gene structural features and relationships of fungal MAC1. (A) Gene structures of PoMAC1a and PoMAC1b in P. ostreatus CCMSSC 00389. (B) A neighbor-joining phylogenetic tree and motifs of 15 MAC1 protein sequences from fungal species.

Figure 2

Amino acid sequence alignment and gene structure diagram of PoMAC1a and PoMAC1b. (A) Amino acid sequence alignment. The Cu-fist motifs of PoMAC1 are enclosed in red rectangles, while the REP-I and II motifs are enclosed in blue rectangles. (B) The PoMAC1 protein molecular peptide chain and the distribution of various functions. The gray area represents the Cu-fist motif, while the red and blue areas represent the REP-I and II motifs, respectively. The listed amino acid sequences are Cu-fist, REP-I, and II motifs.

Figure 3

The subcellular localization of PoMAC1a and PoMAC1b. (A) Prediction of NLS for PoMAC1a and PoMAC1b. (B) The subcellular localization results of PoMAC1a and PoMAC1b. GFP (Green Fluorescent Protein), PoMAC1a GFP, and PoMAC1b GFP were expressed in tobacco leaves; bar = 25 μm.

Figure 4

Verification of OE and RNAi strains. (A) Construction method of OE vectors. (B) Construction method of RNAi-silencing vectors. (C) PCR assay of hyg in P. ostreatus mutant strains, WT, and plasmid. (D) qPCR analysis of the relative expression of PoMAC1 in the tested strains. Different letters indicate significant differences for the comparison of samples (p < 0.05 according to Duncan’s test).

Figure 5

Detection of PoMAC1 mutant strains’ tolerance to 32 °C heat stress. (A) Colony morphology of different strains at 32 °C. (B) The growth rate of different strains at 32 °C. Different letters indicate significant differences for the comparison of samples (p < 0.05 according to Duncan’s test).

Figure 6

PoMAC1 participates in the resistance of mycelia to 40 °C heat stress. (A) The expression levels of PoMAC1a and PoMAC1b under 40 °C stress at different times. (B) The effect of PoMAC1a mutant strains on the recovery growth rate of mycelia after heat stress. (C) The effect of PoMAC1b mutant strains on the recovery growth rate of mycelia after heat stress. The values are the mean ±SE of three independent experiments. Different letters indicate significant differences for the comparison of samples (p < 0.05 according to Duncan’s test).

Figure 7

PoMAC1a promotes primordia formation in P. ostreatus. (A) The expression patterns of PoMAC1a during different developmental stages. (B). The expression patterns of PoMAC1b during different developmental stages. (C) Mushroom production of the WT strain and PoMAC1a mutant strains. (D) Mushroom production of the WT strain and PoMAC1b mutant strains. The values are the mean ± SE of three independent experiments. Different letters indicate significant differences for the comparison of samples (p < 0.05 according to Duncan’s test).