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

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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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VdPbs2 contributes to the oxidative stress response. (A) The ΔVdPbs2 and ΔVdHog1 mutants were compared with the wild type and the ΔVdPbs2/Pbs2 strain. Equal conidial suspension (105 spores/ml) of each strain was sprayed on PDA plates. Sterile filter paper disks with 5 mm diameters were placed in the center of the plates, and 10 μL of 6, 12, and 18 mM H2O2 were added to each paper disk, respectively. The plates were incubated at 28°C for 4 days and the inhibition zones were measured Scale bar = 1 cm. (B) Zones of growth inhibition in (A) were quantified. Error bars represent standard deviation. Asterisk indicates significant difference at P < 0.01. (C) Downregulation of genes related to peroxidase in the ΔVdPbs2 mutant. Relative expression levels of three genes (VDAG_08724, VDAG_03661 and VDAG_06340), which encode peroxidases, were determined by qRT-PCR using the RNA from mycelium treated with 1 mM H2O2 for 30 min. Error bars represent standard deviation.
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Figure 5: VdPbs2 contributes to the oxidative stress response. (A) The ΔVdPbs2 and ΔVdHog1 mutants were compared with the wild type and the ΔVdPbs2/Pbs2 strain. Equal conidial suspension (105 spores/ml) of each strain was sprayed on PDA plates. Sterile filter paper disks with 5 mm diameters were placed in the center of the plates, and 10 μL of 6, 12, and 18 mM H2O2 were added to each paper disk, respectively. The plates were incubated at 28°C for 4 days and the inhibition zones were measured Scale bar = 1 cm. (B) Zones of growth inhibition in (A) were quantified. Error bars represent standard deviation. Asterisk indicates significant difference at P < 0.01. (C) Downregulation of genes related to peroxidase in the ΔVdPbs2 mutant. Relative expression levels of three genes (VDAG_08724, VDAG_03661 and VDAG_06340), which encode peroxidases, were determined by qRT-PCR using the RNA from mycelium treated with 1 mM H2O2 for 30 min. Error bars represent standard deviation.

Mentions: To evaluate the responses of the ΔVdPbs2 mutant to oxidative stress, the inhibition zone was measured on the media containing H2O2. As shown in Figures 5A,B, the ΔVdPbs2 mutant exhibited the larger inhibition zones than the wild type and the ΔVdPbs2/Pbs2 strain at a different concentration of H2O2 suggesting that VdPbs2 is required for H2O2 detoxification. In addition, consistent with our previous observations, loss of VdHog1 did not abolish oxidative sensitivity in V. dahliae (Figure 5A). Furthermore, based on sequence homology, we identified genes encoding H2O2 detoxification in V. dahliae. Transcript analysis revealed that three genes (VDAG_08724, VDAG_03661, and VDAG_06340) were consistently down-regulated in the ΔVdPbs2 mutant compared to that of the wild type after treated with 1 mM H2O2 for 30 min (Figure 5C). Thus, VdPbs2 is essential for the oxidative stress response, but not VdHog1.


The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection
VdPbs2 contributes to the oxidative stress response. (A) The ΔVdPbs2 and ΔVdHog1 mutants were compared with the wild type and the ΔVdPbs2/Pbs2 strain. Equal conidial suspension (105 spores/ml) of each strain was sprayed on PDA plates. Sterile filter paper disks with 5 mm diameters were placed in the center of the plates, and 10 μL of 6, 12, and 18 mM H2O2 were added to each paper disk, respectively. The plates were incubated at 28°C for 4 days and the inhibition zones were measured Scale bar = 1 cm. (B) Zones of growth inhibition in (A) were quantified. Error bars represent standard deviation. Asterisk indicates significant difference at P < 0.01. (C) Downregulation of genes related to peroxidase in the ΔVdPbs2 mutant. Relative expression levels of three genes (VDAG_08724, VDAG_03661 and VDAG_06340), which encode peroxidases, were determined by qRT-PCR using the RNA from mycelium treated with 1 mM H2O2 for 30 min. Error bars represent standard deviation.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037172&req=5

Figure 5: VdPbs2 contributes to the oxidative stress response. (A) The ΔVdPbs2 and ΔVdHog1 mutants were compared with the wild type and the ΔVdPbs2/Pbs2 strain. Equal conidial suspension (105 spores/ml) of each strain was sprayed on PDA plates. Sterile filter paper disks with 5 mm diameters were placed in the center of the plates, and 10 μL of 6, 12, and 18 mM H2O2 were added to each paper disk, respectively. The plates were incubated at 28°C for 4 days and the inhibition zones were measured Scale bar = 1 cm. (B) Zones of growth inhibition in (A) were quantified. Error bars represent standard deviation. Asterisk indicates significant difference at P < 0.01. (C) Downregulation of genes related to peroxidase in the ΔVdPbs2 mutant. Relative expression levels of three genes (VDAG_08724, VDAG_03661 and VDAG_06340), which encode peroxidases, were determined by qRT-PCR using the RNA from mycelium treated with 1 mM H2O2 for 30 min. Error bars represent standard deviation.
Mentions: To evaluate the responses of the ΔVdPbs2 mutant to oxidative stress, the inhibition zone was measured on the media containing H2O2. As shown in Figures 5A,B, the ΔVdPbs2 mutant exhibited the larger inhibition zones than the wild type and the ΔVdPbs2/Pbs2 strain at a different concentration of H2O2 suggesting that VdPbs2 is required for H2O2 detoxification. In addition, consistent with our previous observations, loss of VdHog1 did not abolish oxidative sensitivity in V. dahliae (Figure 5A). Furthermore, based on sequence homology, we identified genes encoding H2O2 detoxification in V. dahliae. Transcript analysis revealed that three genes (VDAG_08724, VDAG_03661, and VDAG_06340) were consistently down-regulated in the ΔVdPbs2 mutant compared to that of the wild type after treated with 1 mM H2O2 for 30 min (Figure 5C). Thus, VdPbs2 is essential for the oxidative stress response, but not VdHog1.

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