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The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection

View Article: PubMed Central - PubMed

ABSTRACT

Verticillium dahliae, a ubiquitous phytopathogenic fungus, forms resting structures, known as microsclerotia that play crucial roles in Verticillium wilt diseases. VdHog1, a mitogen-activated protein kinase (MAPK), controls microsclerotia formation, virulence, and stress response in V. dahliae. In this study, we present detailed evidence that the conserved upstream component of VdHog1, VdPbs2, is a key regulator of microsclerotia formation, oxidative stress and fungicide response and plant virulence in V. dahliae. We identified VdPbs2, homologous to the yeast MAPK kinase Pbs2. Similar to the VdHog1 deletion mutant, VdPbs2 deletion strains exhibited delayed melanin synthesis and reduced formation of microsclerotia. When exposed to stresses, VdPbs2 mutants were more sensitive than the wild type to osmotic agents and peroxide, but more resistant to inhibitors of cell wall synthesis and some fungicides. Finally, VdPbs2 deletion mutants exhibited reduced virulence on smoke tree and tobacco seedlings. When taken together, we implicate that VdPbs2 and VdHog1 function in a cascade that regulates microsclerotia formation and virulence, but not all VdHog1 dependent functions are VdPbs2 regulated. This study thus provides novel insights into the signal transduction mechanisms that regulate microsclerotia formation and pathogenesis in this fungus.

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Deletion of VdPbs2 impairs fungal growth under osmotic stress with hyphal lysis. (A) Colony morphology of the wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 grown at 25°C for 20 days on CM containing 0.8 M NaCl and 1.2 M sorbitol, respectively. Scale bar = 1 cm. (B) The growth rate of the individual strain on CM under osmotic agents. All assays were performed in triplicate. Error bars represent standard deviations. Asterisk indicates significant difference at P < 0.01. (C) Hyphal morphology of the four above strains treated by 0.8 M NaCl and 1.2 M sorbitol, respectively. Under hyperosmotic conditions, the mycelium of the mutant was deformed. HY = hyphae, CO = conidia, DE = deformity. Scale bar = 10 μm. (D) Expression pattern of VdPbs2-GFP in response to osmotic stress at conidia and hyphae. The conidia and hyphae of ΔVdPbs2/Pbs2GFP strains were treated with 0.8 M NaCl for 2 h compared with that of the wild type. HY = hyphae, CO = conidia. Scale bar = 5 μm. (E) The quantification of images fluorescence correlated with (D) in the ΔVdPbs2/Pbs2GFP strain.
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Figure 3: Deletion of VdPbs2 impairs fungal growth under osmotic stress with hyphal lysis. (A) Colony morphology of the wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 grown at 25°C for 20 days on CM containing 0.8 M NaCl and 1.2 M sorbitol, respectively. Scale bar = 1 cm. (B) The growth rate of the individual strain on CM under osmotic agents. All assays were performed in triplicate. Error bars represent standard deviations. Asterisk indicates significant difference at P < 0.01. (C) Hyphal morphology of the four above strains treated by 0.8 M NaCl and 1.2 M sorbitol, respectively. Under hyperosmotic conditions, the mycelium of the mutant was deformed. HY = hyphae, CO = conidia, DE = deformity. Scale bar = 10 μm. (D) Expression pattern of VdPbs2-GFP in response to osmotic stress at conidia and hyphae. The conidia and hyphae of ΔVdPbs2/Pbs2GFP strains were treated with 0.8 M NaCl for 2 h compared with that of the wild type. HY = hyphae, CO = conidia. Scale bar = 5 μm. (E) The quantification of images fluorescence correlated with (D) in the ΔVdPbs2/Pbs2GFP strain.

Mentions: The expression of genes involved in melanin biosynthesis in the VdPbs2 mutant. (A) Expression of five melanin related genes (VDAG_03674, VDAG_00190, VDAG_03665, VDAG_03393 and VDAG_00183) during microsclerotia formation. The β-tubulin was used as an internal reference gene. Total RNA was directly extracted from mycelium of the wild type, ΔVdPbs2, and ΔVdPbs2/Pbs2 grown on PDA plates for 8 days. Error bar represents standard deviation. Asterisk indicates significant difference at P < 0.01. (B) Expression patterns of VdPbs2-GFP during microsclerotia development. GFP expression driven by the native promoter of VdPbs was examined using fluorescence microscope. Spores were cultivated in CM liquid for 4 days. MS1-MS4 represents four typical stages during the entire process of microsclerotia formation at 60 (mycelium at the early stage of inflation), 72 (mycelium inflated completely but without melanin accumulation), 96 h (inflated mycelium with the slight accumulation of melanin), and 14 days (inflated mycelium with the massive accumulation of melanin). Scale bar = 10 μm. (C) The quantification of images fluorescence correlated with Figure 3B. The average brightness of image was performed using the Adobe Photoshop software.


The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection
Deletion of VdPbs2 impairs fungal growth under osmotic stress with hyphal lysis. (A) Colony morphology of the wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 grown at 25°C for 20 days on CM containing 0.8 M NaCl and 1.2 M sorbitol, respectively. Scale bar = 1 cm. (B) The growth rate of the individual strain on CM under osmotic agents. All assays were performed in triplicate. Error bars represent standard deviations. Asterisk indicates significant difference at P < 0.01. (C) Hyphal morphology of the four above strains treated by 0.8 M NaCl and 1.2 M sorbitol, respectively. Under hyperosmotic conditions, the mycelium of the mutant was deformed. HY = hyphae, CO = conidia, DE = deformity. Scale bar = 10 μm. (D) Expression pattern of VdPbs2-GFP in response to osmotic stress at conidia and hyphae. The conidia and hyphae of ΔVdPbs2/Pbs2GFP strains were treated with 0.8 M NaCl for 2 h compared with that of the wild type. HY = hyphae, CO = conidia. Scale bar = 5 μm. (E) The quantification of images fluorescence correlated with (D) in the ΔVdPbs2/Pbs2GFP strain.
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Figure 3: Deletion of VdPbs2 impairs fungal growth under osmotic stress with hyphal lysis. (A) Colony morphology of the wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 grown at 25°C for 20 days on CM containing 0.8 M NaCl and 1.2 M sorbitol, respectively. Scale bar = 1 cm. (B) The growth rate of the individual strain on CM under osmotic agents. All assays were performed in triplicate. Error bars represent standard deviations. Asterisk indicates significant difference at P < 0.01. (C) Hyphal morphology of the four above strains treated by 0.8 M NaCl and 1.2 M sorbitol, respectively. Under hyperosmotic conditions, the mycelium of the mutant was deformed. HY = hyphae, CO = conidia, DE = deformity. Scale bar = 10 μm. (D) Expression pattern of VdPbs2-GFP in response to osmotic stress at conidia and hyphae. The conidia and hyphae of ΔVdPbs2/Pbs2GFP strains were treated with 0.8 M NaCl for 2 h compared with that of the wild type. HY = hyphae, CO = conidia. Scale bar = 5 μm. (E) The quantification of images fluorescence correlated with (D) in the ΔVdPbs2/Pbs2GFP strain.
Mentions: The expression of genes involved in melanin biosynthesis in the VdPbs2 mutant. (A) Expression of five melanin related genes (VDAG_03674, VDAG_00190, VDAG_03665, VDAG_03393 and VDAG_00183) during microsclerotia formation. The β-tubulin was used as an internal reference gene. Total RNA was directly extracted from mycelium of the wild type, ΔVdPbs2, and ΔVdPbs2/Pbs2 grown on PDA plates for 8 days. Error bar represents standard deviation. Asterisk indicates significant difference at P < 0.01. (B) Expression patterns of VdPbs2-GFP during microsclerotia development. GFP expression driven by the native promoter of VdPbs was examined using fluorescence microscope. Spores were cultivated in CM liquid for 4 days. MS1-MS4 represents four typical stages during the entire process of microsclerotia formation at 60 (mycelium at the early stage of inflation), 72 (mycelium inflated completely but without melanin accumulation), 96 h (inflated mycelium with the slight accumulation of melanin), and 14 days (inflated mycelium with the massive accumulation of melanin). Scale bar = 10 μm. (C) The quantification of images fluorescence correlated with Figure 3B. The average brightness of image was performed using the Adobe Photoshop software.

View Article: PubMed Central - PubMed

ABSTRACT

Verticillium dahliae, a ubiquitous phytopathogenic fungus, forms resting structures, known as microsclerotia that play crucial roles in Verticillium wilt diseases. VdHog1, a mitogen-activated protein kinase (MAPK), controls microsclerotia formation, virulence, and stress response in V. dahliae. In this study, we present detailed evidence that the conserved upstream component of VdHog1, VdPbs2, is a key regulator of microsclerotia formation, oxidative stress and fungicide response and plant virulence in V. dahliae. We identified VdPbs2, homologous to the yeast MAPK kinase Pbs2. Similar to the VdHog1 deletion mutant, VdPbs2 deletion strains exhibited delayed melanin synthesis and reduced formation of microsclerotia. When exposed to stresses, VdPbs2 mutants were more sensitive than the wild type to osmotic agents and peroxide, but more resistant to inhibitors of cell wall synthesis and some fungicides. Finally, VdPbs2 deletion mutants exhibited reduced virulence on smoke tree and tobacco seedlings. When taken together, we implicate that VdPbs2 and VdHog1 function in a cascade that regulates microsclerotia formation and virulence, but not all VdHog1 dependent functions are VdPbs2 regulated. This study thus provides novel insights into the signal transduction mechanisms that regulate microsclerotia formation and pathogenesis in this fungus.

No MeSH data available.


Related in: MedlinePlus