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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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Loss of VdPbs2 increases resistance to cell wall stress. (A) Stress responses of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains on CM containing 20 μg/ml CFW and 50 μg/ml CR, respectively. Images were taken at 3 dpi for CFW and 7 dpi for CR. In all assays, the plates were inoculated with conidial solution of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains. Conidial suspension (105/ml and 106 /ml) of the individual strain were spotted on CM media containing the indicated concentration CFW and CR, Scale bar = 0.5 cm. (B) Relative growth of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains treated by the indicated cell stress. Error bar represents standard deviation. Asterisk indicates significant difference at P < 0.01. (C) The expression of two genes (VDAG_08591 and VDAG_03141) involved in chitin synthesis was increased in the ΔVdPbs2 mutant. Error bars indicate standard deviations derived from three independent experiments consisting of three replicas each.
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Figure 4: Loss of VdPbs2 increases resistance to cell wall stress. (A) Stress responses of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains on CM containing 20 μg/ml CFW and 50 μg/ml CR, respectively. Images were taken at 3 dpi for CFW and 7 dpi for CR. In all assays, the plates were inoculated with conidial solution of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains. Conidial suspension (105/ml and 106 /ml) of the individual strain were spotted on CM media containing the indicated concentration CFW and CR, Scale bar = 0.5 cm. (B) Relative growth of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains treated by the indicated cell stress. Error bar represents standard deviation. Asterisk indicates significant difference at P < 0.01. (C) The expression of two genes (VDAG_08591 and VDAG_03141) involved in chitin synthesis was increased in the ΔVdPbs2 mutant. Error bars indicate standard deviations derived from three independent experiments consisting of three replicas each.

Mentions: To determine whether deletion of VdPbs2 affects the response to cell wall stress in V. dahliae, we tested cell viability of the ΔVdPbs2 mutant under cell wall stressors such as CFW and CR. Conidia (105 conidia/ml and 106 conidia/ml) of ΔVdPbs2, ΔVdHog1, wild type, and ΔVdPbs2/Pbs2 strain were spotted on CM media containing CFW (20 μg/ml) and CR (50 μg/ml), respectively. Enhanced growth on media with CFW (20 μg/ml) and CR (50 μg/ml), respectively, was observed for ΔVdPbs2 and ΔVdHog1 mutants. By contrast, reduced growth was observed for the wild type and ΔVdPbs2/Pbs2 strain (Figures 4A,B), suggesting VdPbs2 and VdHog1 are involved in the response to cell wall stress.


The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection
Loss of VdPbs2 increases resistance to cell wall stress. (A) Stress responses of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains on CM containing 20 μg/ml CFW and 50 μg/ml CR, respectively. Images were taken at 3 dpi for CFW and 7 dpi for CR. In all assays, the plates were inoculated with conidial solution of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains. Conidial suspension (105/ml and 106 /ml) of the individual strain were spotted on CM media containing the indicated concentration CFW and CR, Scale bar = 0.5 cm. (B) Relative growth of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains treated by the indicated cell stress. Error bar represents standard deviation. Asterisk indicates significant difference at P < 0.01. (C) The expression of two genes (VDAG_08591 and VDAG_03141) involved in chitin synthesis was increased in the ΔVdPbs2 mutant. Error bars indicate standard deviations derived from three independent experiments consisting of three replicas each.
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Figure 4: Loss of VdPbs2 increases resistance to cell wall stress. (A) Stress responses of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains on CM containing 20 μg/ml CFW and 50 μg/ml CR, respectively. Images were taken at 3 dpi for CFW and 7 dpi for CR. In all assays, the plates were inoculated with conidial solution of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains. Conidial suspension (105/ml and 106 /ml) of the individual strain were spotted on CM media containing the indicated concentration CFW and CR, Scale bar = 0.5 cm. (B) Relative growth of wild type, ΔVdPbs2, ΔVdPbs2/Pbs2, and ΔVdHog1 strains treated by the indicated cell stress. Error bar represents standard deviation. Asterisk indicates significant difference at P < 0.01. (C) The expression of two genes (VDAG_08591 and VDAG_03141) involved in chitin synthesis was increased in the ΔVdPbs2 mutant. Error bars indicate standard deviations derived from three independent experiments consisting of three replicas each.
Mentions: To determine whether deletion of VdPbs2 affects the response to cell wall stress in V. dahliae, we tested cell viability of the ΔVdPbs2 mutant under cell wall stressors such as CFW and CR. Conidia (105 conidia/ml and 106 conidia/ml) of ΔVdPbs2, ΔVdHog1, wild type, and ΔVdPbs2/Pbs2 strain were spotted on CM media containing CFW (20 μg/ml) and CR (50 μg/ml), respectively. Enhanced growth on media with CFW (20 μg/ml) and CR (50 μg/ml), respectively, was observed for ΔVdPbs2 and ΔVdHog1 mutants. By contrast, reduced growth was observed for the wild type and ΔVdPbs2/Pbs2 strain (Figures 4A,B), suggesting VdPbs2 and VdHog1 are involved in the response to cell wall stress.

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