Verticillium dahliae causes destructive vascular wilt diseases on more than 200 plant species, including economically important crops and ornamental trees worldwide. The melanized microsclerotia enable the fungus to survive for years in soil and are crucial for its disease cycle. Previously, we found that the VdPbs2-VdHog1 (V. dahliae Pbs2-V. dahliae Hog1) module plays key roles in microsclerotial formation, stress responses, and virulence in V. dahliae. In this study, two mitogen-activated protein kinase kinase kinases (MAPKKKs) homologous to Ssk2p and Ste11p, which activate the Pbs2p-Hog1p module by phosphorylation in budding yeast, were identified in the genome of V. dahliae. Both ΔVdSsk2 (V. dahliae Ssk2) and ΔVdSte11 strains showed severe defects in microsclerotial formation and melanin biosynthesis, but the relative importance of these two genes in microsclerotial development was different. Deletion of VdSsk2, but not VdSte11, affected responses to osmotic stress, fungicidal response, and cell wall stressors. The ΔVdSsk2 strain exhibited a significant reduction in virulence, while the ΔVdSte11 strain was nonpathogenic due to failure to penetrate and form hyphopodia. Phosphorylation assays demonstrated that VdSsk2, but not VdSte11, can phosphorylate VdHog1 in V. dahliae. Moreover, VdCrz1, encoding a calcineurin-responsive zinc finger transcription factor and a key regulator of calcium signaling in fungi, was misregulated in the ΔVdSsk2, ΔVdPbs2, and ΔVdHog1 mutants.
IMPORTANCE These data provide insights into the distinctive functions of VdSsk2 and VdSte11 in pathogenicity, stress adaptation, and microsclerotial formation in V. dahliae.
The fungus Verticillium dahliae causes vascular wilt disease on hundreds of plant species. Homologs of the bZIP transcription factor Atf1 are required for virulence in most pathogenic fungi, but the molecular basis for their involvement is largely unknown.We performed targeted gene deletion, expression analysis, biochemistry and pathogenicity assays to demonstrate that VdAtf1 governs pathogenesis via the regulation of nitrosative resistance and nitrogen metabolism in V. dahliae.VdAtf1 controls pathogenesis via the regulation of nitric oxide (NO) resistance and inorganic nitrogen metabolism rather than oxidative resistance and is important for penetration peg formation in V. dahliae. VdAtf1 affects ammonium and nitrate assimilation in response to various nitrogen sources. VdAtf1 may be involved in regulating the expression of VdNut1. VdAtf1 responds to NO stress by strengthening the fungal cell wall, and by causing over-accumulation of methylglyoxal and glycerol, which in turn impacts NO detoxification. We also verified that the VdAtf1 ortholog in Fusarium graminearum mediates nitrogen metabolism, suggesting conservation of this function in related plant pathogenic fungi.Our findings revealed new functions of VdAtf1 in pathogenesis, response to nitrosative stress and nitrogen metabolism in V. dahliae. The results provide novel insights into the regulatory mechanisms of the transcription factor VdAtf1 in virulence.
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