The Gal4-Type Transcription Factor Pro1 Integrates Inputs from Two Different MAPK Cascades to Regulate Development in the Fungal Pathogen Fusarium oxysporum

Abstract

Mitogen-activated protein kinase (MAPK) signaling pathways control fundamental aspects of growth and development in fungi. In the soil-inhabiting ascomycete Fusarium oxysporum, which causes vascular wilt disease in more than a hundred crops, the MAPKs Fmk1 and Mpk1 regulate an array of developmental and virulence-related processes. The downstream components mediating these disparate functions are largely unknown. Here we find that the GATA-type transcription factor Pro1 integrates signals from both MAPK pathways to control a subset of functions, including quorum sensing, hyphal fusion and chemotropism. By contrast, Pro1 is dispensable for other downstream processes such as invasive hyphal growth and virulence, or response to cell wall stress. We further show that regulation of Pro1 activity by these upstream pathways occurs at least in part at the level of transcription. Besides the MAPK pathways, upstream regulators of Pro1 transcription also include the Velvet regulatory complex, the signaling protein Soft (Fso1) and the transcription factor Ste12 which was previously shown to act downstream of Fmk1. Collectively, our results reveal a role of Pro1 in integrating the outputs from different signaling pathways of F. oxysporum thereby mediating key developmental decisions in this important fungal pathogen.

Keywords: Fmk1; Fusarium oxysporum; MAPK; Mpk1; Pro1; hyphal fusion; transcription factor; virulence.

Figure 1

Transcriptional analysis of pro1 in different F. oxysporum strains. Quantitative real-time RT-PCR analysis was performed in the indicated strains germinated for 10 h in Puhalla minimal medium supplemented with 25 mM sodium glutamate and 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid and adjusted to pH 7.4. Transcript levels of the pro1 gene are expressed relative to those of the wild type strain (wt). Data show means ± s.d. **, p < 0.001; ***, p < 0.0001 versus wt according to t test with Welch’s correction.

Figure 2

Pro1 is required for vegetative hyphal fusion and mycelial aggregation. (A) Microconidia of the indicated strains were germinated on water agar plates supplemented with 25 mM NaNO₃. After 15 h the percentage of germ tubes undergoing vegetative hyphal fusion was determined. Data show means ± s.d. Columns with the same letter are not significantly different according to one-way ANOVA followed by Tukey’s multiple comparison test (p < 0.05). (B) Formation of hyphal aggregates after 36 h growth in minimal medium supplemented with 25 mM NaNO₃. Fungal cultures were vortexed to dissociate weakly adhered hyphae, transferred to a multiwell plate and imaged under a Lumar V12 stereomicroscope equipped with an AxioCam MR5 camera.

Figure 3

Pro1 controls quorum sensing of F. oxysporum microconidia. The percentage of germinated microconidia of the indicated strains at optimal (3.2 × 10⁵ mL⁻¹) (A) or inhibitory microconidial concentrations (8.6 × 10⁷ mL⁻¹) (B) was determined after 13 or 15 h, respectively, of incubation in minimal medium supplemented with 0.1% (w/v) sucrose and adjusted to pH 5.0. Data show means ± s.d. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 versus wt according to one-way ANOVA followed by Tukey’s multiple comparison test.

Figure 4

Pro1 is required for hyphal chemotropism towards nutrients (A), sex pheromone (B) and plant roots (C). Directed growth of germ tubes of the indicated strains towards a gradient of indicated chemoattractants was determined. Data show means ± s.d. *, p < 0.05; **, p < 0.01 versus wt according to Yates’ corrected chi-squared test (two-sided).

Figure 5

Pro1 is dispensable for invasive hyphal growth and virulence of F. oxysporum on tomato plants. (A) Cellophane penetration assay. The indicated strains were spot-inoculated on top of a cellophane membrane on a PDA plate, grown for 3 days at 28 °C and imaged (Before). The cellophane with the fungal colony was removed and plates were incubated for an additional day to determine the presence of mycelial growth on the plate, indicating penetration of the cellophane (After). (B) Tomato root infection assay. Kaplan–Meier plot showing survival of groups of 10 tomato plants (cv. Monica) inoculated by dipping roots into a suspension of 5 × 10⁶ microconidia/mL of the indicated fungal strains. Percentage survival was plotted for 30 days (****, p < 0.0001). Data shown are from one representative experiment. All experiments were performed at least three times with similar results.

Figure 6

Diagram showing regulation of Pro1 by different upstream components in F. oxysporum.