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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

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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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Sensitivity of the WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) to oxidative stress.(A) 4, 2, and 1 μl conidial suspensions prepared from WT, ΔClVelB, and ClVelB-C were dripped on a CM plate with the with the indicated concentrations of H2O2. ΔClVelB is more sensitive to H2O2 than WT and ClVelB-C. (B) qRT-PCR analysis of the catalase-encoding gene cat3. 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 a T-test analysis. The expression levels of cat3 were reduced in ΔClVelB (3.7-fold at time 0 and 3.3-fold 30 min after H2O2 addition).
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f8: Sensitivity of the WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) to oxidative stress.(A) 4, 2, and 1 μl conidial suspensions prepared from WT, ΔClVelB, and ClVelB-C were dripped on a CM plate with the with the indicated concentrations of H2O2. ΔClVelB is more sensitive to H2O2 than WT and ClVelB-C. (B) qRT-PCR analysis of the catalase-encoding gene cat3. 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 a T-test analysis. The expression levels of cat3 were reduced in ΔClVelB (3.7-fold at time 0 and 3.3-fold 30 min after H2O2 addition).

Mentions: The growth rates of the mutants were quantified on media supplemented with stressors that induce osmotic stress (1.2 M NaCl, 1.2 M KCl), fungicides (10 μg/mL iprodione, 0.1 μg/mL fludioxonil), and oxidative stress (2.0 or 4.0 mM H2O2) to assess whether ClVelB is also essential to cope with various kinds of stresses. Under osmotic stress conditions, all mutants showed comparable growth rates. ΔClVelB showed a slightly decreased resistance to osmotic stresses cultured in 1.2 M NaCl or 1.2 M KCl medium and a slight decreased sensitivity to the dicarboximide fungicide iprodione and phenylpyrrole fungicide fludioxonil (Fig. 6). The intercellular glycerol of fungus plays a significant role in responding to osmotic stress11. As shown in Fig. 7, ΔClVelB exhibited a low basal level of glycerol accumulation and the expression of the gpd1 gene that is responsible for glycerol synthesis showed a similar trend, which partially explains why ΔClVelB exhibited decreased resistance to osmotic stresses. ΔClVelB showed high sensitivity to H2O2 compared to the WT strain, reintroduction of WT clvelB gene into the mutant restored the tolerance of WT to oxidative stress (Fig. 8A).


Involvement of a velvet protein ClVelB in the regulation of vegetative differentiation, oxidative stress response, secondary metabolism, and virulence in Curvularia lunata
Sensitivity of the WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) to oxidative stress.(A) 4, 2, and 1 μl conidial suspensions prepared from WT, ΔClVelB, and ClVelB-C were dripped on a CM plate with the with the indicated concentrations of H2O2. ΔClVelB is more sensitive to H2O2 than WT and ClVelB-C. (B) qRT-PCR analysis of the catalase-encoding gene cat3. 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 a T-test analysis. The expression levels of cat3 were reduced in ΔClVelB (3.7-fold at time 0 and 3.3-fold 30 min after H2O2 addition).
© Copyright Policy - open-access
Related In: Results  -  Collection

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f8: Sensitivity of the WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) to oxidative stress.(A) 4, 2, and 1 μl conidial suspensions prepared from WT, ΔClVelB, and ClVelB-C were dripped on a CM plate with the with the indicated concentrations of H2O2. ΔClVelB is more sensitive to H2O2 than WT and ClVelB-C. (B) qRT-PCR analysis of the catalase-encoding gene cat3. 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 a T-test analysis. The expression levels of cat3 were reduced in ΔClVelB (3.7-fold at time 0 and 3.3-fold 30 min after H2O2 addition).
Mentions: The growth rates of the mutants were quantified on media supplemented with stressors that induce osmotic stress (1.2 M NaCl, 1.2 M KCl), fungicides (10 μg/mL iprodione, 0.1 μg/mL fludioxonil), and oxidative stress (2.0 or 4.0 mM H2O2) to assess whether ClVelB is also essential to cope with various kinds of stresses. Under osmotic stress conditions, all mutants showed comparable growth rates. ΔClVelB showed a slightly decreased resistance to osmotic stresses cultured in 1.2 M NaCl or 1.2 M KCl medium and a slight decreased sensitivity to the dicarboximide fungicide iprodione and phenylpyrrole fungicide fludioxonil (Fig. 6). The intercellular glycerol of fungus plays a significant role in responding to osmotic stress11. As shown in Fig. 7, ΔClVelB exhibited a low basal level of glycerol accumulation and the expression of the gpd1 gene that is responsible for glycerol synthesis showed a similar trend, which partially explains why ΔClVelB exhibited decreased resistance to osmotic stresses. ΔClVelB showed high sensitivity to H2O2 compared to the WT strain, reintroduction of WT clvelB gene into the mutant restored the tolerance of WT to oxidative stress (Fig. 8A).

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