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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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ClVelB regulates cell wall integrity.(A) Sensitivity of the WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) to the cell wall damaging agents. The detection was made on a CM plate added with the corresponding cell wall damaging agent. (B) Expression changes of gls2 and slt2 in each strain. The relative expression levels of gls2 and slt2 in ΔClVelB are the relative cDNA amounts of the same gene in the WT strain. Line bars indicate the standard errors from the three trial replicates. A single asterisk indicates a p-value < 0.05 while double asterisks indicate a p-value < 0.001 in the T-test analysis. (C) Ultrastructural analyses of the cell of the clvelB deletion mutant. Cells of the WT and ΔClVelB were observed with a transmission electronic microscope (Tecnai G2 Spirit Biotwin, FEI). Mycelia were harvested and fixed in glutaraldehyde for 12 h at 4 °C.
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f9: ClVelB regulates cell wall integrity.(A) Sensitivity of the WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) to the cell wall damaging agents. The detection was made on a CM plate added with the corresponding cell wall damaging agent. (B) Expression changes of gls2 and slt2 in each strain. The relative expression levels of gls2 and slt2 in ΔClVelB are the relative cDNA amounts of the same gene in the WT strain. Line bars indicate the standard errors from the three trial replicates. A single asterisk indicates a p-value < 0.05 while double asterisks indicate a p-value < 0.001 in the T-test analysis. (C) Ultrastructural analyses of the cell of the clvelB deletion mutant. Cells of the WT and ΔClVelB were observed with a transmission electronic microscope (Tecnai G2 Spirit Biotwin, FEI). Mycelia were harvested and fixed in glutaraldehyde for 12 h at 4 °C.

Mentions: The deletion of clvelB led to a decrease in resistance to osmotic stresses, which indicates that ClVelB might regulate the integrity of the cell wall and/or the cell member. To prove this hypothesis, we tested the sensitivity of ΔClVelB to cell wall damaging agents, namely, Congo red and Caffeine and to cell member damaging agent SDS. The results indicate that ΔClVelB displayed a decreased resistance to these compounds to some extent (Fig. 9A). Studies have shown that Congo red could disturb the fungal cell wall by binding to cellulose and chitin12. Thus, we tested the expressions of the 1,3-beta-glucan synthase gene gls2 and MAPK gene slt2, which are homologous to the core element genes of Saccharomyces cerevisiae cell wall integrity (CWI) pathway, in the clvelb deletion mutant. The expression levels of gls2 and slt2 in ΔClVelB were lower than those in WT (Fig. 9B), which agrees with the phenotype that ΔClVelB showed decreased resistance to Congo red. More interestingly, we found that the deletion of clvelB led to the decrease of fungal cell width compared with WT (Fig. 9C). These results demonstrate that ClVelB might be related to the regulation of the CWI pathway in C. lunata.


Involvement of a velvet protein ClVelB in the regulation of vegetative differentiation, oxidative stress response, secondary metabolism, and virulence in Curvularia lunata
ClVelB regulates cell wall integrity.(A) Sensitivity of the WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) to the cell wall damaging agents. The detection was made on a CM plate added with the corresponding cell wall damaging agent. (B) Expression changes of gls2 and slt2 in each strain. The relative expression levels of gls2 and slt2 in ΔClVelB are the relative cDNA amounts of the same gene in the WT strain. Line bars indicate the standard errors from the three trial replicates. A single asterisk indicates a p-value < 0.05 while double asterisks indicate a p-value < 0.001 in the T-test analysis. (C) Ultrastructural analyses of the cell of the clvelB deletion mutant. Cells of the WT and ΔClVelB were observed with a transmission electronic microscope (Tecnai G2 Spirit Biotwin, FEI). Mycelia were harvested and fixed in glutaraldehyde for 12 h at 4 °C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5385503&req=5

f9: ClVelB regulates cell wall integrity.(A) Sensitivity of the WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) to the cell wall damaging agents. The detection was made on a CM plate added with the corresponding cell wall damaging agent. (B) Expression changes of gls2 and slt2 in each strain. The relative expression levels of gls2 and slt2 in ΔClVelB are the relative cDNA amounts of the same gene in the WT strain. Line bars indicate the standard errors from the three trial replicates. A single asterisk indicates a p-value < 0.05 while double asterisks indicate a p-value < 0.001 in the T-test analysis. (C) Ultrastructural analyses of the cell of the clvelB deletion mutant. Cells of the WT and ΔClVelB were observed with a transmission electronic microscope (Tecnai G2 Spirit Biotwin, FEI). Mycelia were harvested and fixed in glutaraldehyde for 12 h at 4 °C.
Mentions: The deletion of clvelB led to a decrease in resistance to osmotic stresses, which indicates that ClVelB might regulate the integrity of the cell wall and/or the cell member. To prove this hypothesis, we tested the sensitivity of ΔClVelB to cell wall damaging agents, namely, Congo red and Caffeine and to cell member damaging agent SDS. The results indicate that ΔClVelB displayed a decreased resistance to these compounds to some extent (Fig. 9A). Studies have shown that Congo red could disturb the fungal cell wall by binding to cellulose and chitin12. Thus, we tested the expressions of the 1,3-beta-glucan synthase gene gls2 and MAPK gene slt2, which are homologous to the core element genes of Saccharomyces cerevisiae cell wall integrity (CWI) pathway, in the clvelb deletion mutant. The expression levels of gls2 and slt2 in ΔClVelB were lower than those in WT (Fig. 9B), which agrees with the phenotype that ΔClVelB showed decreased resistance to Congo red. More interestingly, we found that the deletion of clvelB led to the decrease of fungal cell width compared with WT (Fig. 9C). These results demonstrate that ClVelB might be related to the regulation of the CWI pathway in C. 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 &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