FgMet3 and FgMet14 related to cysteine and methionine biosynthesis regulate vegetative growth, sexual reproduction, pathogenicity, and sensitivity to fungicides in Fusarium graminearum

Abstract

Fusarium graminearum is a destructive filamentous fungus, which widely exists in wheat and other cereal crops. Cysteine and Methionine are unique sulfur-containing amino acids that play an essential role in protein synthesis and cell life, but their functions and regulation in F. graminearum remain largely unknown. Here we identified two proteins, FgMet3 and FgMet14 in F. graminearum, which are related to the synthesis of cysteine and methionine. We found FgMet3 and FgMet14 were localized to the cytoplasm and there was an interaction between them. FgMet3 or FgMet14 deletion mutants (ΔFgMet3 and ΔFgMet14) were deficient in vegetative growth, pigment formation, sexual development, penetrability and pathogenicity. With exogenous addition of cysteine and methionine, the vegetative growth and penetrability could be completely restored in ΔFgMet3 and ΔFgMet14, while sexual reproduction could be fully restored in ΔFgMet3 and partially restored in ΔFgMet14. ΔFgMet3 and ΔFgMet14 exhibited decreased sensitivity to Congo red stress and increased sensitivity to SDS, NaCl, KCl, Sorbitol, Menadione, and Zn ion stresses. Moreover, FgMet3 and FgMet14 nonspecifically regulate the sensitivity of F. graminearum to fungicides. In conclusion, FgMet3 and FgMet14 interacted to jointly regulate the development, pathogenicity, pigment formation, sensitivity to fungicides and stress factors in F. graminearum.

Keywords: FgMet14; FgMet3; cysteine; fungicide sensitivity; fusarium graminearum; methionine; pathogenicity; sexual reproduction.

Figure 1

FgMet3 interacts with FgMet14. Co-immunoprecipitation (Co-IP) assay confirmed the interaction between FgMet3 and FgMet14.

Figure 2

Vegetative growth of PH-1, ΔFgMet3, and ΔFgMet14 mutants and their complements on different mediums. (A) The colonial morphology of strains on PDA, CM, MM, V8 medium at 25°C for 3 days. (B) Hyphal growth rate of the strains on PDA, CM, MM, V8 medium. Error bars in each column denote standard error of three repetitions. Within each treatment, bars with the same letter indicate no significant difference according to the least significant difference (LSD) test at p = 0.05.

Figure 3

Colony diameter and morphology of PH-1, ΔFgMet3 and ΔFgMet14 mutants and their complements cultured on MM medium supplemented with different concentrations (1μg/mL, 10μg/mL and 40μg/mL) of cysteine and methionine. The strains were cultured on the medium at 25°C for 3 days.

Figure 4

The sensitivity of ΔFgMet3 and ΔFgMet14 to different fungicides compared with PH-1 and complements. (A) Effects of 0.5 μg/mL carbendazim, 0.03125 μg/mL fludioxonil, 0.2 μg/mL tebuconazole, 0.01 μg/mL pydiflumetofen, 4 μg/mL cyprodinil, 0.1 μg/mL fluoxastrobin, 0.2 μg/mL phenamacril, 0.4 μg/mL chlorothalonil, 0.5 μg/mL prothioconazole and 0.1 μg/mL fluazinam on mycelial linear growth of ΔFgMet3 and ΔFgMet14 mutants. (B) The inhibition of the mycelial growth rate was examined after each strain was incubated for 3 days on PDA supplemented with each fungicide. Bars denote standard deviations from three experiments. Within each treatment, the same letter indicate no significant difference according to the least significant difference (LSD) test. P = 0.05.

Figure 5

The sensitivity of ΔFgMet3 and ΔFgMet14 to different stress agents compared with PH-1 and complements. (A) The sensitivity of Fusarium graminearum wild-type PH-1, knockout mutants, and complementation strains to 500 µg/mL Congo red, 0.03% SDS, 1 M NaCl, 1 M KCl, 1.5 M Sorbitol, 5 mM ZnCl₂, and 0.1 mM Menadione on PDA plate. (B) The inhibition of the mycelial growth rate was examined after each strain was incubated for 3 days on PDA supplemented with each stress compound. Bars denote standard deviations from three experiments. Within each treatment, bars with the same letter indicate no significant difference according to the least significant difference (LSD) test. P = 0.05.

Figure 6

Pigment formation of PH-1, FgMet3 or FgMet14 deletion mutants and their complements. (A) All the strains were cultured on PDA medium for 2d and cultured in PDB medium for 3d to produce pigment. (B) Relative expression levels of Aurofusarin biosynthesis-related genes FgAurJ, FgAurZ, FgAurF, and FgAurO in the ΔFgMet3 and ΔFgMet14 mutants compared with the WT strain. Bars with the same letter indicate no significant difference according to the least significant difference (LSD) test. P = 0.05.

Figure 7

Sexual reproduction of PH-1, FgMet3 or FgMet14 deletion mutants and their complements. (A) The sexual reproduction of strains when incubated on carrot medium for 7 days. (B) The sexual reproduction of strains when incubated on carrot medium for 11 days. (C) The sexual reproduction of strains when incubated on carrot medium for 15 days. (D) Relative expression levels of sexual reproduction-related genes Mat1-1-1, Mat1-1-2, Mat1-1-3, and Mat1-2-1 in the ΔFgMet3 and ΔFgMet14 mutants compared with the WT strain. Bars with the same letter indicate no significant difference according to the least significant difference (LSD) test. P = 0.05.

Figure 8

Exogenous addition of cysteine and methionine could partially restore the sexual reproductive ability of FgMet3 and FgMet14 deletion mutants. (A) The sexual reproduction of strains when incubated on carrot medium which contains 40µg/mL cysteine or methionine for 7 days. (B) The sexual reproduction of strains when incubated on carrot medium which contains 40µg/mL cysteine or methionine for 11 days.

Figure 9

Defects of ΔFgMet3 and ΔFgMet14 mutants in plant infection. (A) Wheat coleoptiles were examined at 10 days after inoculated with a conidial suspension. (B) Mean spot length of the strains. Bars with the same letter indicate no significant difference at p = 0.05. (C) Flowering wheat heads were examined at 14 days post-inoculation (dpi). The inoculation sites were indicated by black dots. (D, E) The tomato mean spot diameter were examined at 3 days after placed with fresh dishes. (F) FgMet3 and FgMet14 are not important for DON biosynthesis of Fusarium graminearum. All strains were incubated in GYEP for 7 days and mycelia were harvested and dry for weighing. (G) FgMet3 and FgMet14 didn’t regulate TRI5, TRI6 gene expression in Fusarium graminearum. All strains were incubated in GYEP for 36 h and mycelia were harvested for qRT-PCR. Bars with the same letter indicate no significant difference according to the least significant difference (LSD) test at p = 0.05.

Figure 10

Defects of ΔFgMet3 and ΔFgMet14 mutants in plant penetration. (A) The penetration of strains. The sterilized double-layer cellophane was placed on PDA, CM, MM plates, and the MM plates containing 40 µg/mL methionine or cysteine for this experiment. (B) The penetration rate of strains. The penetration rate (%) = mean diameter after penetration/mean diameter before penetration × 100. Bars with the same letter indicate no significant difference according to the least significant difference (LSD) test at p = 0.05.

Figure 11

Subcellular localization of FgMet3 and FgMet14. FgMet3 and FgMet14 were localized to the cytoplasm. All strains were incubated in YEPD for 12-24 h and MBL for 3 d to obtain mycelium and conidia.

Figure 12

A proposed model showing the function of FgMet3 and FgMet14. FgMet3 interacts with FgMet14 indirectly, and they regulate development, pathogenicity, pigment formation, sensitivity to fungicides and stress factors of F. graminearum.