MaCts1, an Endochitinase, Is Involved in Conidial Germination, Conidial Yield, Stress Tolerances and Microcycle Conidiation in Metarhizium acridum

Abstract

Entomopathogenic fungi are promising biocontrol agents of insect-mediated crop damage. Microcycle conidiation has shown great potential in enhancing the conidial yield and quality of entomopathogenic fungi. Homologs of Cts1, an endochitinase of Saccharomyces cerevisiae, participate in cell separation in several fungal spp. and may contribute to the morphological differences that occur during the shift to microcycle conidiation. However, the precise functions of Cts1 in entomopathogenic fungi remain unclear. Herein, the endochitinase gene, MaCts1, was characterized in the model entomopathogen, Metarhizium acridum. A loss of function line for MaCts1 led to a delay of 1 h in the median germination time, a 28% reduction in conidial yield and significant defects in fungal resistances to UV-irradiation (18%) and heat-shock (15%), while fungal tolerances to cell wall stressors, oxidative and hyperosmotic stresses and virulence remained unchanged. The MaCts1-disruption strain displayed typical conidiation on the microcycle conidiation induction medium, SYA. In contrast, deletion of key genes in the morphogenesis-related NDR kinase network (MOR pathway)/regulation of Ace2 and morphogenesis (RAM pathway) did not affect the SYA-induction of microcycle conidiation. This indicates that MaCts1 makes contributions to the microcycle conidiation, which may not be dependent on the MOR/RAM pathway in M. acridum.

Keywords: Cts1; MOR/RAM pathway; endochitinase; entomopathogenic fungus; microcycle conidiation.

Figure 1

Structural and phylogenetic features of MaCts1. (A) Domain structure analysis of MaCts1. Glyco_hydro_18 is a glycosyl hydrolase family 18 domain and CBM_1 is a cellulose binding domain. (B) Phylogenetic analysis of Cts1 protein sequences from different fungi. The sequences used were Metarhizium acridum CQMa102, XP_007814832.1; Metarhizium robertsii, EXU99126.1; Metarhizium rileyi, TWU71928.1; Beauveria bassiana, KAH8712633.1; Cordyceps militaris, ATY58486.1; Aspergillus nidulans, CBF74135.1; Saccharomyces cerevisiae, NP_013388.1; Candida albicans, EAL00460.1; Puccinia graminis, XP_003327072.1; Melampsora larici, XP_007410264.1; Ustilago maydis, XP_011390771.1; Moesziomyces antarcticus, XP_014654591.1, Suillus luteus (KIK43475.1).

Figure 2

Inactivation of MaCts1 impaired conidial germination and influenced conidial yields, but not virulence of M. acridum. (A) The conidial germination (%) of the WT, ΔMaCts1 and CP strains examined on 1/4 SDAY medium. (B) The median germination time (GT₅₀). (C) The conidial yields of the WT, ΔMaCts1 and CP strains assessed on 1/4 SDAY medium. (D) The initial stage of conidiation of the WT, ΔMaCts1 and CP strains on 1/4 SDAY medium. Arrows indicate conidia on the conidiophores. (E) Locust survival (%) after topical inoculation of conidia from the different strains. (F) The median lethal times (LT₅₀s). Error bars indicate the standard deviation from triplicate experiments. One, two and three asterisks indicate a significant difference at p < 0.05, p < 0.01, p < 0.001, respectively. “ns” indicates no significant difference.

Figure 3

Stress tolerance assays of the WT, ΔMaCts1 and CP strains. (A) The effect of UV-B irradiation on conidial germination (%) after UV-B irradiation followed by 20 h of cultivation on 1/4 SDAY medium at 28 °C. (B) The median inhibition times (IT₅₀s) of fungal strains treated by UV-B irradiation. (C) The effect of heat shock (44.5 °C) on conidial germination. (D) IT₅₀s of fungal strains treated by heat-shock (44.5 °C). (E) Fungal colonies on 1/4 SDAY alone or supplemented with sorbitol (SOR; 1 mol/L), calcofluor white (CFW;50 μg/mL), SDS (0.01% w/v), NaCl (1 mol/L), congo red (CR; 500 μg/mL), menadione (0.07% w/v) and SDS (0.01% w/v), respectively. Error bars indicate the standard deviation from triplicate experiments. One and two asterisks indicate a significant difference at p < 0.05 and p < 0.01, respectively. “ns” indicates no significant difference.

Figure 4

The conidiation pattern and hyphal morphology. (A) The conidiation pattern of the WT, ΔMaCts1 and CP strains on SYA medium. (B) The hyphae from the WT, ΔMaCts1 and CP strains grown on SYA medium for 18, 24, 30 h. The hyphae were stained with calcofluor white. Arrows indicate mycelial septa. (C) The length of apical hyphal cells in the WT, ΔMaCts1 and CP strains. (D) The length of sub-apical hyphal cells in the WT, ΔMaCts1 and CP strains. Error bars indicate standard deviations from triplicate experiments. Three asterisks indicate significant differences at p < 0.001, one asterisks indicate significant differences at p < 0.05 and “ns” indicates no difference.

Figure 5

The key components of the MOR/RAM pathway are not related with the shift in conidiation pattern in M. acridum. The conidiation pattern of WT and disrupted mutants at 18 h and 24 h after incubation on SYA medium.