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

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.

Keywords: MAP kinase pathway; Verticillium dahliae; microsclerotia formation; pathogenicity; stress responses.

FIGURE 1

Loss of VdPbs2 leads to reduced microsclerotia formation. (A) Colony morphology of the wild type, ΔVdPbs2, ΔVdPbs2/Pbs2 and ΔVdHog1 grown on PDA for 8 days. The inset shows colony from the opposite view. (B) Microsclerotia formation of the individual strain on cellulose membrane placed onto basal medium plates, and incubated at 25°C at 3, 5, and 24 days. Conidia from each strain were sprayed on the cellulose membrane at a concentration of 10⁵ conidia/ml. (C) Microscopic observation of microsclerotia formation of the above four strains at 24 dpi. Scale bar = 1 mm. (D) Melanized area fractions in the colony were counted by ImageJ. Asterisk indicates significant difference at P < 0.01.

FIGURE 2

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.

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.

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.

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.

FIGURE 6

VdPbs2 deletion mutants exhibit distinct responses to different fungicides. (A) The ΔVdPbs2 and ΔVdHog1 mutants showed enhanced resistance to fludioxonil and iprodione. Conidial suspension (10⁵/ml and 10⁶ /ml) of the wild type, ΔVdPbs2 and ΔVdHog1, and the the ΔVdPbs2/Pbs2 were spotted on CM media with the indicated concentration of fludioxonil and iprodione, respectively. (B) The ΔVdPbs2 and ΔVdHog1 mutants exhibited more sensitivity to chlorothalonil and difenoconazole. Conidial suspension (10⁵/ml and 10⁶ /ml) of the above strains were spotted on CM media with the indicated concentration of chlorothalonil and difenoconazole, respectively. (C) Growth rate of the above strains on CM containing with the indicated concentration of fludioxonil and iprodione, respectively. Error bar represents standard deviation. Asterisk indicates significant difference at P < 0.01.

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.