Limits...
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.

No MeSH data available.


Related in: MedlinePlus

ClVelB negatively regulates the mycelial melanization of C. lunata.(A) Bottom of the CM plates of WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) grown in constant light (LL) or dark (DD) for 7 days. Photos were taken after removing conidia. Note the heavy melanization of mycelia of ΔClVelB in both LL and DD compared to WT. (B) The mycelial pellet of WT, ΔClVelB, and ClVelB-C at different indicated time points. ΔClVelB is melanized by 60 h, which is ahead of WT and ClVelB-C. Pigmentation starts by 68 h in WT and ClVelB-C. (C) qRT-PCR analyses of pks18, cmr1, brn1, brn2, and scd. Expression was tested at 48 and 60 h. The expression level compared with the WT at 48 h is shown. Error bars are the standard deviation. A single asterisk indicates the p-value < 0.05 in a T-test analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5385503&req=5

f4: ClVelB negatively regulates the mycelial melanization of C. lunata.(A) Bottom of the CM plates of WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) grown in constant light (LL) or dark (DD) for 7 days. Photos were taken after removing conidia. Note the heavy melanization of mycelia of ΔClVelB in both LL and DD compared to WT. (B) The mycelial pellet of WT, ΔClVelB, and ClVelB-C at different indicated time points. ΔClVelB is melanized by 60 h, which is ahead of WT and ClVelB-C. Pigmentation starts by 68 h in WT and ClVelB-C. (C) qRT-PCR analyses of pks18, cmr1, brn1, brn2, and scd. Expression was tested at 48 and 60 h. The expression level compared with the WT at 48 h is shown. Error bars are the standard deviation. A single asterisk indicates the p-value < 0.05 in a T-test analysis.

Mentions: Similar to Botrytis cinerea BcVelB, the deletion of ClVelB leads to an increase in mycelial pigmentation8, and insufficient ClVelB leads to the increased melanization of mycelia, which is grown both on a solid (Fig. 4A) and in a liquid CM medium (Fig. 4B), indicating that ClVelB negatively regulates the mycelial pigmentation in C. lunata. Hyphal pigmentation develops faster in ΔClVelB than in WT (Fig. 4B). By 68 h, all strains were darkly pigmented. We detected the expression of the PKS gene (pks18), the transcription factor gene (cmr1), and three synthase genes (brn1, brn2, and scd) related to the synthesis of DHN melanin in the WT and mutant to further confirm this observation (Fig. 4C)49. qRT-PCR analyses showed that the expression levels of pks18 in ΔClVelB were enhanced compared to those in WT. By 48 h, the expression of pks18 in ΔClVelB has a 12.53-fold increase, which peaked at 60 h (57.82-fold). At 48 h, the expression of cmr1 has a 5.25-fold increase in ΔClVelB compared to that in WT. brn1, brn2, and scd also showed high expression levels in ΔClVelB compared to those in WT at both 48 and 60 h. For all the genes, the reintroduction of clvelB restored the WT expression levels. Overall, we conclude that ClVelB plays a negative regulation role in the synthesis of melanin. The pyroquilon and kojic acid inhibitors were used to study the influence on melanization and support our previous studies that the conidial and mycelial melanin of C. lunata is not the tyrosine-derived but DHN type10. While the colors of all the cultures (WT, ΔClVelB, and ClVelB-C) grown on kojic acid remained the same, those grown on pyroquilon were changing from black to light brown (Fig. 5), bolstering previous research on melanization 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 negatively regulates the mycelial melanization of C. lunata.(A) Bottom of the CM plates of WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) grown in constant light (LL) or dark (DD) for 7 days. Photos were taken after removing conidia. Note the heavy melanization of mycelia of ΔClVelB in both LL and DD compared to WT. (B) The mycelial pellet of WT, ΔClVelB, and ClVelB-C at different indicated time points. ΔClVelB is melanized by 60 h, which is ahead of WT and ClVelB-C. Pigmentation starts by 68 h in WT and ClVelB-C. (C) qRT-PCR analyses of pks18, cmr1, brn1, brn2, and scd. Expression was tested at 48 and 60 h. The expression level compared with the WT at 48 h is shown. Error bars are the standard deviation. A single asterisk indicates the p-value < 0.05 in a T-test analysis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: ClVelB negatively regulates the mycelial melanization of C. lunata.(A) Bottom of the CM plates of WT strain (CX-3), clvelB deletion mutant (ΔClVelB), and complemented strain (ClVelB-C) grown in constant light (LL) or dark (DD) for 7 days. Photos were taken after removing conidia. Note the heavy melanization of mycelia of ΔClVelB in both LL and DD compared to WT. (B) The mycelial pellet of WT, ΔClVelB, and ClVelB-C at different indicated time points. ΔClVelB is melanized by 60 h, which is ahead of WT and ClVelB-C. Pigmentation starts by 68 h in WT and ClVelB-C. (C) qRT-PCR analyses of pks18, cmr1, brn1, brn2, and scd. Expression was tested at 48 and 60 h. The expression level compared with the WT at 48 h is shown. Error bars are the standard deviation. A single asterisk indicates the p-value < 0.05 in a T-test analysis.
Mentions: Similar to Botrytis cinerea BcVelB, the deletion of ClVelB leads to an increase in mycelial pigmentation8, and insufficient ClVelB leads to the increased melanization of mycelia, which is grown both on a solid (Fig. 4A) and in a liquid CM medium (Fig. 4B), indicating that ClVelB negatively regulates the mycelial pigmentation in C. lunata. Hyphal pigmentation develops faster in ΔClVelB than in WT (Fig. 4B). By 68 h, all strains were darkly pigmented. We detected the expression of the PKS gene (pks18), the transcription factor gene (cmr1), and three synthase genes (brn1, brn2, and scd) related to the synthesis of DHN melanin in the WT and mutant to further confirm this observation (Fig. 4C)49. qRT-PCR analyses showed that the expression levels of pks18 in ΔClVelB were enhanced compared to those in WT. By 48 h, the expression of pks18 in ΔClVelB has a 12.53-fold increase, which peaked at 60 h (57.82-fold). At 48 h, the expression of cmr1 has a 5.25-fold increase in ΔClVelB compared to that in WT. brn1, brn2, and scd also showed high expression levels in ΔClVelB compared to those in WT at both 48 and 60 h. For all the genes, the reintroduction of clvelB restored the WT expression levels. Overall, we conclude that ClVelB plays a negative regulation role in the synthesis of melanin. The pyroquilon and kojic acid inhibitors were used to study the influence on melanization and support our previous studies that the conidial and mycelial melanin of C. lunata is not the tyrosine-derived but DHN type10. While the colors of all the cultures (WT, ΔClVelB, and ClVelB-C) grown on kojic acid remained the same, those grown on pyroquilon were changing from black to light brown (Fig. 5), bolstering previous research on melanization 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