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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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Reduced virulence of the VdPbs2 mutant on smoke tree and tobacco seedlings. (A) One-year-old smoke trees were inoculated with conidia concentration of 106/ml of the wild type and ΔVdPbs2 mutant. The pictures were taken at 45 dpi. Twenty seedlings were inoculated with each strain. (B) Two-month-old tobacco seedlings were inoculated with the same methods mentioned in (A). The assays were performed in triplicate. The pictures were taken at 40 dpi. (C) Height of tobacco seedlings inoculated with the above strains. The height of tobacco seedlings measured at 30 dpi. (D) The mortality of smoke tree (at 30, 45 dpi) and tobacco (at 20, 40 dpi) inoculated with the wild type, ΔVdPbs2 and ΔVdPbs2/Pbs2 strains.
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Figure 7: Reduced virulence of the VdPbs2 mutant on smoke tree and tobacco seedlings. (A) One-year-old smoke trees were inoculated with conidia concentration of 106/ml of the wild type and ΔVdPbs2 mutant. The pictures were taken at 45 dpi. Twenty seedlings were inoculated with each strain. (B) Two-month-old tobacco seedlings were inoculated with the same methods mentioned in (A). The assays were performed in triplicate. The pictures were taken at 40 dpi. (C) Height of tobacco seedlings inoculated with the above strains. The height of tobacco seedlings measured at 30 dpi. (D) The mortality of smoke tree (at 30, 45 dpi) and tobacco (at 20, 40 dpi) inoculated with the wild type, ΔVdPbs2 and ΔVdPbs2/Pbs2 strains.

Mentions: We next sought to address whether VdPbs2 plays a role in virulence in plants. We used seedlings of smoke tree and tobacco to carry out the virulence experiments. On both hosts, the ΔVdPbs2 mutant exhibited striking reduced virulence (Figures 7A,B) and only less 20% mortality of plants at 45 dpi (Figure 7D). By contrast, at 45 dpi, up to 80% mortality of which inoculated with the wild type and the ΔVdPbs2/Pbs2 strain showed clear wilt symptoms, including chlorosis (Figures 7A,B) and obviously reduced plant height (Figures 7B,C). Due to the limitations, we just further observed the penetration of the strain on onion epidermis. The wild type could infect epidermal cells and expand into the epidermal tissues, whereas the ΔVdPbs2 mutant hardly infects epidermal cells even though the mutants produced long germ tubes (Supplementary Figure S4). Together, these results indicated that VdPbs2 may be involved in the penetration process during plant infection.


The Mitogen-Activated Protein Kinase Kinase VdPbs2 of Verticillium dahliae Regulates Microsclerotia Formation, Stress Response, and Plant Infection
Reduced virulence of the VdPbs2 mutant on smoke tree and tobacco seedlings. (A) One-year-old smoke trees were inoculated with conidia concentration of 106/ml of the wild type and ΔVdPbs2 mutant. The pictures were taken at 45 dpi. Twenty seedlings were inoculated with each strain. (B) Two-month-old tobacco seedlings were inoculated with the same methods mentioned in (A). The assays were performed in triplicate. The pictures were taken at 40 dpi. (C) Height of tobacco seedlings inoculated with the above strains. The height of tobacco seedlings measured at 30 dpi. (D) The mortality of smoke tree (at 30, 45 dpi) and tobacco (at 20, 40 dpi) inoculated with the wild type, ΔVdPbs2 and ΔVdPbs2/Pbs2 strains.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5037172&req=5

Figure 7: Reduced virulence of the VdPbs2 mutant on smoke tree and tobacco seedlings. (A) One-year-old smoke trees were inoculated with conidia concentration of 106/ml of the wild type and ΔVdPbs2 mutant. The pictures were taken at 45 dpi. Twenty seedlings were inoculated with each strain. (B) Two-month-old tobacco seedlings were inoculated with the same methods mentioned in (A). The assays were performed in triplicate. The pictures were taken at 40 dpi. (C) Height of tobacco seedlings inoculated with the above strains. The height of tobacco seedlings measured at 30 dpi. (D) The mortality of smoke tree (at 30, 45 dpi) and tobacco (at 20, 40 dpi) inoculated with the wild type, ΔVdPbs2 and ΔVdPbs2/Pbs2 strains.
Mentions: We next sought to address whether VdPbs2 plays a role in virulence in plants. We used seedlings of smoke tree and tobacco to carry out the virulence experiments. On both hosts, the ΔVdPbs2 mutant exhibited striking reduced virulence (Figures 7A,B) and only less 20% mortality of plants at 45 dpi (Figure 7D). By contrast, at 45 dpi, up to 80% mortality of which inoculated with the wild type and the ΔVdPbs2/Pbs2 strain showed clear wilt symptoms, including chlorosis (Figures 7A,B) and obviously reduced plant height (Figures 7B,C). Due to the limitations, we just further observed the penetration of the strain on onion epidermis. The wild type could infect epidermal cells and expand into the epidermal tissues, whereas the ΔVdPbs2 mutant hardly infects epidermal cells even though the mutants produced long germ tubes (Supplementary Figure S4). Together, these results indicated that VdPbs2 may be involved in the penetration process during 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.

No MeSH data available.


Related in: MedlinePlus