Modification and functional adaptation of the MBF1 gene family in the lichenized fungus Endocarpon pusillum under environmental stress

Abstract

The multiprotein-bridging factor 1 (MBF1) gene family is well known in archaea, non-lichenized fungi, plants, and animals, and contains stress tolerance-related genes. Here, we identified four unique mbf1 genes in the lichenized fungi Endocarpon spp. A phylogenetic analysis based on protein sequences showed the translated MBF1 proteins of the newly isolated mbf1 genes formed a monophyletic clade different from other lichen-forming fungi and Ascomycota groups in general, which may reflect the evolution of the biological functions of MBF1s. In contrast to the lack of function reported in yeast, we determined that lysine¹¹⁴ in the deduced Endocarpon pusillum MBF1 protein (EpMBF1) had a specific function that was triggered by environmental stress. Further, the Endocarpon-specific C-terminus of EpMBF1 was found to participate in stress tolerance. Epmbf1 was induced by a number of abiotic stresses in E. pusillum and transgenic yeast, and its stress-resistant ability was stronger than that of the yeast mbf1. These findings highlight the evolution and function of EpMBF1 and provide new insights into the co-evolution hypothesis of MBF1 and TATA-box-binding proteins.

Figure 1

Bayesian phylogenetic tree based on the amino acid sequences of MBF1s. The Bayesian tree was constructed with MrBayes 3.2.6 on the CIPRES Science Gateway. The 39 MBF1 sequences represent Archaea, Fungi, Animalia, and Planta (see Supplementary Table S2). Different colours indicate the different groups. The numbers at each node represent posterior probability values. Numbers greater than 0.90 are shown above branches. Scale bar = 0.2 substitutions per site.

Figure 2

Analysis of EpMBF1 in vivo and in vitro. (A) Expression patterns of Epmbf1 in E. pusillum exposed to different stresses. The abiotic stress treatments were 0.5 mM NaCl, 10 mM H₂O₂, and 20% PEG. The expression level in control medium (CK) was considered as the reference. (B) Subcellular distribution of the EpMBF1-GFP fusion protein. Scale bar = 5 μm. (C) Interactions between yTBP and MBF1s of E. pusillum and S. cerevisiae in vitro as determined by the GST pull-down assay. (D) Semi-quantitative RT-PCR assay to confirm the results of the ymbf1 gene knock down. pda1 was used as the reference gene, and its expression level was adjusted and then compared with the expression level of ymbf1 in the mutant and WT strains. Cropped images are shown; the full length gel and blots are included in Supplementary Information.

Figure 3

Schematic overview of the EpMBF1–yMBF1 chimeric proteins. (A) Original amino acid sequences of yMBF1 and EpMBF1, with different colours indicating the different functional parts. (B) Rearranged amino acid sequences used to construct the EpMBF1–yMBF1 chimeric proteins.

Figure 4

Effects of MBF1s and the chimeric proteins on oxidative and heat stress tolerance in yeast mutants. Yeast strains grown in a concentration grade on selective plates with no treatment (CK), 2.5 mM H₂O₂, and 51.5 h at 45 °C, respectively. Photos were taken after 72 h at 30 °C.

Figure 5

Expression analysis of stress-related genes in transgenic yeast under control and stress conditions. pda1 was used as the reference gene, and Δmbf1/pyes2 was used as the reference sample. Asterisks indicate a significant difference (*P < 0.001) compared with the reference sample (CK). Genes with more than twofold differential expressions are listed with coloured labels.