Functional analysis of a novel endo-β-1,6-glucanase Mo Glu16 and its application in detecting cell wall β-1,6-glucan of Magnaporthe oryzae

Abstract

As an essential component of the fungal cell wall, β-1,6-glucan has an important role in the growth and development of fungi, but its distribution has not been investigated in Magnaporthe oryzae. Here, a novel β-1,6-glucanase from M. oryzae, MoGlu16, was cloned and expressed in Pichia pastoris. The enzyme was highly active on pustulan, with a specific activity of 219.0 U/mg at pH 5.0 and 50°C, and showed great selectivity for continuous β-1,6-glycosidic bonding polysaccharides. Based on this, β-1,6-glucan was selectively visualized in the vegetative hyphae, conidia and bud tubes of M. oryzae using a hydrolytically inactive GFP-tagged MoGlu16 with point mutations at the catalytic position (His-MoGlu16^E236A-Gfp). The spore germination and appressorium formation were significantly inhibited after incubation of 10⁵/ml conidia with 0.03 μg/μl MoGlu16. Mycelia treated with MoGlu16 produced reactive oxygen species and triggered the cell wall integrity pathway, increasing the expression levels of genes involved in cell wall polysaccharide synthesis. These results revealed that MoGlu16 participated in the remodeling of cell wall in M. oryzae, laying a foundation for the analysis of cell wall structure.

Keywords: Magnaporthe oryzae; cell wall; specificity; β-1,6-glucan; β-1,6-glucanase.

Figure 1

Enzymatic characteristics of MoGlu16. (A) Influence of pH on the activity of MoGlu16 with pustulan as the substrate at 50°C. The solid line represented the pH profile and the dotted line represented the pH stability of MoGlu16. (B) Impact of temperature on the activity of MoGlu16 with pustulan as the substrate at pH 5.0. The solid line represented the temperature profile and the dotted line represented the thermostability of MoGlu16. (C) Effect of the substrate concentration of pustulan on the hydrolytic activity of MoGlu16. Bars show the standard deviations of the averages from three replicates.

Figure 2

Analysis of the hydrolysates after the action of MoGlu16 to pustulan. (A) TLC analysis of the hydrolysates produced at different incubation time (5 min, 15 min, 30 min, 1 h, 2 h, 4 h, 8 h and 12 h). (B) MALDI-TOF MS spectra of the reaction mixture of pustulan and MoGlu16 at 12 h.

Figure 3

Construction of the fluorescent derivatives derived from MoGlu16 without hydrolytic activity. (A) Hydrolytic activity analysis of MoGlu16 and its derivatives (MoGlu16^E236A and MoGlu16^E332A) to pustulan. Bars indicate the standard deviations of the averages from three replicates. Columns with different letters are significantly different at p ≤ 0.05 according to Duncan’s test. (B) Construction and expression of the different fluorescent derivatives. (1) and (2), the construction of the derived proteins His-MoGlu16^E236A-Gfp and MoGlu16^E236A-Gfp-His, respectively; (3), the expression of the derivatives by SDS-PAGE detection. (C) Fluorescence detection of MoGlu16 and the derivatives (His-MoGlu16^E236A-Gfp and MoGlu16^E236A-Gfp-His) under blue light.

Figure 4

Localization of β-1,6-glucan in different development stages of M. oryzae. The cells of M. oryzae (hyphae, conidia, germ tube and appressoria) were incubated, respectively, with His-MoGlu16^E236A-Gfp for 1 h at 28°C, and then washed with 20 mM Tris–HCl buffer. Finally, the cells were observed under fluorescence microscope. LFM, light-field microscope; FM, fluorescence microscope. All images were observed at a magnification of 63 under confocal laser scanning microscopy (CLSM) (CLSM, Leisa, TCSSP2). The results represent one of three replicates with similar results.

Figure 5

Effect of MoGlu16 on conidial development. (A) Assay of conidial germination treated by various concentrations of MoGlu16. Conidial germination (4 h) and appressorium formation (8 h) were observed by differential interference contrast (DIC) microscope. (B) Quantitative analysis of the conidial germination and the appressorium formation. (C) Assay of conidial germination treated by 0.03 μg/μl inactivated MoGlu16. Bars indicate the standard deviations of the averages from three replicates.

Figure 6

Effects of MoGlu16 on mycelial growth of M. oryzae. (A,B) Detection of ROS accumulation in mycelial apical (A) and median (B) based on H₂DCFDA staining after treatment with MoGlu16 for 12 h. LFM, light-field microscope; FM, fluorescence microscope; Guy11, untreated M. oryzae; Guy11-IMoGlu16, M. oryzae treated by 0.30 μg/mL inactivatd MoGlu16; Guy11-MoGlu16, M. oryzae treated by 0.30 μg/mL MoGlu16. (C) Detection of cell membrane integrity of mycelium via PI staining after treatment with MoGlu16 for 12 h. (D) Detection of chitin in the cell wall of mycelium treated by MoGlu16 for 12 h. (E) Expression levels of genes related to reactive oxygen species production. These genes contained glyoxalase gene (MGG_02069), peroxidase genes (MGG_04404, MGG_04545 and MGG_13239), superoxide-generating NADPH-oxidase genes (NOX1, MGG_00750; and NOX2, MGG_06599) and oxidative stress regulator gene (YAP1, MGG_12814). (F) Expression levels of genes correlated with cell wall polysaccharide synthases. These genes included chitin synthase genes (CHS1, MGG_01802; CHS2, MGG_04245; CHS3, MGG_09551; CHS4, MGG_09962; CHS5, MGG_13014; CHS6, MGG_13013; and CHS7, MGG_06064), β-1,3-glucan synthase gene FKS1 (MGG_00865) and α-1,3-glucan synthase gene AGS2 (MGG_09639). The values represent the means of the three replicates with standard deviation (error bars). Columns with different letters are significantly different at p ≤ 0.05 according to Duncan’s test.