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Involvement of a velvet protein ClVelB in the regulation of vegetative differentiation, oxidative stress response, secondary metabolism, and virulence in Curvularia lunata

View Article: PubMed Central - PubMed

ABSTRACT

The ortholog of Aspergillus nidulans VelB, which is known as ClVelB, was studied to gain a broader insight into the functions of a velvet protein in Curvularia lunata. With the expected common and specific functions of ClVelB, the deletion of clvelB results in similar though not identical phenotypes. The pathogenicity assays revealed that ΔClVelB was impaired in colonizing the host tissue, which corresponds to the finding that ClVelB controls the production of conidia and the methyl 5-(hydroxymethyl) furan-2-carboxylate toxin in C. lunata. However, the deletion of clvelB led to the increase in aerial hyphae and melanin formation. In addition, ΔClVelB showed a decreased sensitivity to iprodione and fludioxonil fungicides and a decreased resistance to cell wall-damaging agents and osmotic stress and tolerance to H2O2. The ultrastructural analysis indicated that the cell wall of ΔClVelB became thinner, which agrees with the finding that the accumulated level of glycerol in ΔClVelB is lower than the wild-type. Furthermore, the interaction of ClVelB with ClVeA and ClVosA was identified in the present research through the yeast two-hybrid and bimolecular fluorescence complementation assays. Results indicate that ClVelB plays a vital role in the regulation of various cellular processes in C. lunata.

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Related in: MedlinePlus

Effects of ClVelB on colony morphology and sporulation.(A) Cultures grown on CM plates under constant light (LL) or dark (DD), and 12 h light/dark cycle (LD) conditions for 7 days at 28 °C. Note that in LL, WT and ClVelB-C are white and flat, while ΔClVelB is pigmented and fluffy, which reflects aerial hyphal growth. Alternating banding rhythm in the middle plate suggests that the conidiation of WT is responsive to light. This banding rhythm is greatly reduced in ΔClVelB. (B) Side view of the plates of WT, ΔClVelB, and ClVelB-C grown in LL or DD on CM. Note the aerial hyphae on the plates of ΔClVelB, especially from LL. By contrast, the surface of the WT and ClVelB-C only shows a few aerial hyphae. (C) Quantification of conidia from cultures grown under LL, LD, and DD conditions. Error bars are the standard deviation. A single asterisk indicates the p-value < 0.05 while double asterisks indicate the p-value < 0.001 in the T-test analysis. Sporulation of ΔClVelB is repressed in all circumstances, while this was not observed for WT and ClVelB-C. (D) Hyphae structures of WT and ΔClVelB were examined through scanning electron microscopy (Sirion 200, FEI).
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f3: Effects of ClVelB on colony morphology and sporulation.(A) Cultures grown on CM plates under constant light (LL) or dark (DD), and 12 h light/dark cycle (LD) conditions for 7 days at 28 °C. Note that in LL, WT and ClVelB-C are white and flat, while ΔClVelB is pigmented and fluffy, which reflects aerial hyphal growth. Alternating banding rhythm in the middle plate suggests that the conidiation of WT is responsive to light. This banding rhythm is greatly reduced in ΔClVelB. (B) Side view of the plates of WT, ΔClVelB, and ClVelB-C grown in LL or DD on CM. Note the aerial hyphae on the plates of ΔClVelB, especially from LL. By contrast, the surface of the WT and ClVelB-C only shows a few aerial hyphae. (C) Quantification of conidia from cultures grown under LL, LD, and DD conditions. Error bars are the standard deviation. A single asterisk indicates the p-value < 0.05 while double asterisks indicate the p-value < 0.001 in the T-test analysis. Sporulation of ΔClVelB is repressed in all circumstances, while this was not observed for WT and ClVelB-C. (D) Hyphae structures of WT and ΔClVelB were examined through scanning electron microscopy (Sirion 200, FEI).

Mentions: Target gene deletion strategy was employed by replacing clvelB with a hygromycin resistance (hph) cassette to investigate the biological functions of ClVelB in C. lunata (Fig. 2). The Southern hybridization pattern confirmed that homologous recombination occurs at the clvelB locus in ΔClVelB. Complementation of the deletion mutant (ClVelB-C) was accomplished by the reintroduction of wild-type (WT) clvelB into the genome of ΔClVelB. The radial growth rates of the mutants and WT on the complete medium (CM) under different light conditions (constant light [LL] or dark [DD], and 12 hours of light/dark photoperiod [LD]) were compared. ΔClVelB had a significantly slower mycelial growth rate than WT and the complemented strain ClVelB-C on the CM medium (Table 1). As the primary source of inoculum for host infections, conidia are formed during exposure to light. Time course experiments were performed to follow the onset of conidiation in the generated mutant under different illumination conditions. WT and ClVelB-C exhibited an obvious banding rhythm which reflected periods of conidiation under LD conditions, whereas that in ΔClVelB was greatly reduced (Fig. 3A). The conidiation of WT was the most in the LL condition, the least in the DD condition, and a moderate number in the LD condition (Fig. 3C). However, the conidiation of ΔClVelB sharply declined, and the differences in conidiation in the preceding three conditions were not as obvious as in WT and ClVelB-C (Fig. 3C).


Involvement of a velvet protein ClVelB in the regulation of vegetative differentiation, oxidative stress response, secondary metabolism, and virulence in Curvularia lunata
Effects of ClVelB on colony morphology and sporulation.(A) Cultures grown on CM plates under constant light (LL) or dark (DD), and 12 h light/dark cycle (LD) conditions for 7 days at 28 °C. Note that in LL, WT and ClVelB-C are white and flat, while ΔClVelB is pigmented and fluffy, which reflects aerial hyphal growth. Alternating banding rhythm in the middle plate suggests that the conidiation of WT is responsive to light. This banding rhythm is greatly reduced in ΔClVelB. (B) Side view of the plates of WT, ΔClVelB, and ClVelB-C grown in LL or DD on CM. Note the aerial hyphae on the plates of ΔClVelB, especially from LL. By contrast, the surface of the WT and ClVelB-C only shows a few aerial hyphae. (C) Quantification of conidia from cultures grown under LL, LD, and DD conditions. Error bars are the standard deviation. A single asterisk indicates the p-value < 0.05 while double asterisks indicate the p-value < 0.001 in the T-test analysis. Sporulation of ΔClVelB is repressed in all circumstances, while this was not observed for WT and ClVelB-C. (D) Hyphae structures of WT and ΔClVelB were examined through scanning electron microscopy (Sirion 200, FEI).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5385503&req=5

f3: Effects of ClVelB on colony morphology and sporulation.(A) Cultures grown on CM plates under constant light (LL) or dark (DD), and 12 h light/dark cycle (LD) conditions for 7 days at 28 °C. Note that in LL, WT and ClVelB-C are white and flat, while ΔClVelB is pigmented and fluffy, which reflects aerial hyphal growth. Alternating banding rhythm in the middle plate suggests that the conidiation of WT is responsive to light. This banding rhythm is greatly reduced in ΔClVelB. (B) Side view of the plates of WT, ΔClVelB, and ClVelB-C grown in LL or DD on CM. Note the aerial hyphae on the plates of ΔClVelB, especially from LL. By contrast, the surface of the WT and ClVelB-C only shows a few aerial hyphae. (C) Quantification of conidia from cultures grown under LL, LD, and DD conditions. Error bars are the standard deviation. A single asterisk indicates the p-value < 0.05 while double asterisks indicate the p-value < 0.001 in the T-test analysis. Sporulation of ΔClVelB is repressed in all circumstances, while this was not observed for WT and ClVelB-C. (D) Hyphae structures of WT and ΔClVelB were examined through scanning electron microscopy (Sirion 200, FEI).
Mentions: Target gene deletion strategy was employed by replacing clvelB with a hygromycin resistance (hph) cassette to investigate the biological functions of ClVelB in C. lunata (Fig. 2). The Southern hybridization pattern confirmed that homologous recombination occurs at the clvelB locus in ΔClVelB. Complementation of the deletion mutant (ClVelB-C) was accomplished by the reintroduction of wild-type (WT) clvelB into the genome of ΔClVelB. The radial growth rates of the mutants and WT on the complete medium (CM) under different light conditions (constant light [LL] or dark [DD], and 12 hours of light/dark photoperiod [LD]) were compared. ΔClVelB had a significantly slower mycelial growth rate than WT and the complemented strain ClVelB-C on the CM medium (Table 1). As the primary source of inoculum for host infections, conidia are formed during exposure to light. Time course experiments were performed to follow the onset of conidiation in the generated mutant under different illumination conditions. WT and ClVelB-C exhibited an obvious banding rhythm which reflected periods of conidiation under LD conditions, whereas that in ΔClVelB was greatly reduced (Fig. 3A). The conidiation of WT was the most in the LL condition, the least in the DD condition, and a moderate number in the LD condition (Fig. 3C). However, the conidiation of ΔClVelB sharply declined, and the differences in conidiation in the preceding three conditions were not as obvious as in WT and ClVelB-C (Fig. 3C).

View Article: PubMed Central - PubMed

ABSTRACT

The ortholog of Aspergillus nidulans VelB, which is known as ClVelB, was studied to gain a broader insight into the functions of a velvet protein in Curvularia lunata. With the expected common and specific functions of ClVelB, the deletion of clvelB results in similar though not identical phenotypes. The pathogenicity assays revealed that &Delta;ClVelB was impaired in colonizing the host tissue, which corresponds to the finding that ClVelB controls the production of conidia and the methyl 5-(hydroxymethyl) furan-2-carboxylate toxin in C. lunata. However, the deletion of clvelB led to the increase in aerial hyphae and melanin formation. In addition, &Delta;ClVelB showed a decreased sensitivity to iprodione and fludioxonil fungicides and a decreased resistance to cell wall-damaging agents and osmotic stress and tolerance to H2O2. The ultrastructural analysis indicated that the cell wall of &Delta;ClVelB became thinner, which agrees with the finding that the accumulated level of glycerol in &Delta;ClVelB is lower than the wild-type. Furthermore, the interaction of ClVelB with ClVeA and ClVosA was identified in the present research through the yeast two-hybrid and bimolecular fluorescence complementation assays. Results indicate that ClVelB plays a vital role in the regulation of various cellular processes in C. lunata.

No MeSH data available.


Related in: MedlinePlus