The vtc4 gene influences polyphosphate storage, morphogenesis, and virulence in the maize pathogen Ustilago maydis

Abstract

The maize pathogen Ustilago maydis switches from budding to filamentous, dikaryotic growth in response to environmental signals including nutrient status, growth in the host, and the presence of mating pheromones. The filamentous dikaryon is capable of proliferating within host tissue to cause disease symptoms including tumors. The transition from yeast cells to hyphal filaments is regulated by a mitogen-activated protein kinase cascade and a cyclic-AMP-protein kinase A (PKA) pathway. Serial analysis of gene expression with PKA mutants identified orthologs of components of the PHO phosphate acquisition pathway as transcriptional targets of the PKA pathway, and these included genes for Pho84, an acid phosphatase, and the vacuolar transport chaperones Vtc1 and Vtc4. In Saccharomyces cerevisiae, Vtc4p is required during the fusion of inorganic-phosphate-containing vesicles to the vacuolar membrane and the consequent accumulation of phosphate stored as polyphosphate (polyP) in the vacuole. We found that deletion of vtc4 in U. maydis also reduced polyP stored in vacuoles. Intriguingly, Deltavtc4 mutants possessed a filamentous cellular morphology, in contrast to the budding, yeast-like growth of the wild-type parent. The Deltavtc4 mutants also displayed decreased symptom development and reduced proliferation in planta. The interaction with PKA signaling was further investigated by the generation of Deltavtc4 ubc1 double mutants. Deletion of vtc4 completely suppressed the multiple-budded phenotype of a Deltaubc1 mutant, indicating that polyP stores are essential for this PKA-induced trait. Overall, this study reveals a novel role for PKA-regulated polyP accumulation in the control of fungal morphogenesis and virulence.

FIG. 1.

Strains with the Δvtc4 allele exhibit a filamentous colonial morphology. Wild-type (A) and Δvtc4 (B) strains were grown for 5 days at 30°C on MM plus 1% glucose and 1, 7.35, or 250 mM KH₂PO₄ (pH 7). Colonies are shown at ×2 and ×6 magnifications. (A) Wild-type colonies exhibit a smooth, uniform colonial morphology. (B) Colonies of Δvtc4 mutant strains have a filamentous morphology in high concentrations of KH₂PO₄. Scale bars = 1 mm.

FIG. 2.

The Δvtc4 mutants have a filamentous cellular morphology in a high phosphate concentration. Wild-type (A) or Δvtc4 (B) cells were grown in MM plus 1% glucose and 1, 7.35, or 250 mM KH₂PO₄ (pH 7). (A) Wild-type haploid U. maydis cells grow as budding yeast regardless of the extracellular phosphate concentration. (B) Δvtc4 cells grow predominately as budding yeast cells in 1 mM KH₂PO₄. In contrast, the majority of Δvtc4 cells grown in 7.35 mM KH₂PO₄ appear as elongated cells. At high phosphate concentrations (250 mM), the Δvtc4 strains grow predominately as septate, branched hyphal filaments. Images were captured by DIC or epifluorescence microscopy to observe CAL-stained cell walls. Scale bars = 20 μm.

FIG. 3.

The Δvtc4 strains lack accumulated vacuolar polyP at high concentrations of extracellular phosphate. Wild-type (A) or Δvtc4 (B) cells were grown in MM plus 1% glucose and 1, 7.35, or 250 mM KH₂PO₄ (pH 7). Cells were stained with Toluidine Blue O to observe polyP. (A) Wild-type cells grown in 1 mM KH₂PO₄ do not show polyP accumulated in the vacuole. In contrast, wild-type cells grown in either 7.35 or 250 mM KH₂PO₄ show large accumulations of polyP in the vacuole. polyP accumulations are indicated by arrowheads. (B) Similar to wild-type cells, Δvtc4 cells grown in 1 mM KH₂PO₄ grow as budding cells which lack polyP accumulation in the vacuole. In contrast to wild-type cells, Δvtc4 cells grown in 7.35 mM KH₂PO₄ produce elongated cells which lack polyP accumulated in the vacuole. Likewise, in 250 mM KH₂PO₄, Δvtc4 cells exhibit a filamentous morphology and lack vacuole polyP accumulation. Images were captured by DIC microscopy. Scale bars = 20 μm. (C) polyP accumulation in wild-type and Δvtc4 cells after overnight growth in MM plus 1, 7.35, or 250 mM KH₂PO₄. A 20% polyacrylamide gel was loaded with 10 μg of total RNA of each sample and 20 μg of polyP ladder (P25 or P45 for the major polyP chain length). polyP was stained with Toluidine Blue O. The increasing amounts of polyP in wild-type extracts correlate with increasing concentrations of KH₂PO₄. In contrast, only a slight accumulation of polyP is visible in extracts from Δvtc4 cells grown in 1 mM or 7.35 mM KH₂PO₄ and no accumulation of polyP is visible in extracts from the Δvtc4 mutant grown in 250 mM KH₂PO₄.

FIG. 4.

Mating is unaffected in Δvtc4 strains. (A) To assess mating capability, cultures of wild-type or Δvtc4 cells were cospotted onto CM-plus-charcoal plates and incubated for 2 days at 30°C. Drops of single wild-type and mutant strains were included as controls. A positive mating reaction is indicated by a fuzzy colony morphology. Combinations of mutant strains show that deletion of vtc4 does not reduce the ability to mate. (B) To examine mating filaments, cells were scraped from CM-plus-charcoal plates and resuspended in sdH₂O. Wild-type cells responding to pheromone extend conjugation tubes that fuse to form a filamentous dikaryon. The Δvtc4 strains also extend conjugation tubes that fuse to form mating filaments. Images were captured by DIC microscopy. Scale bars = 20 μm.

FIG. 5.

Teliospore production is unaffected in Δvtc4 strains. (A) The developing ears of 2-month-old maize plants were inoculated with crosses of wild-type strains 518 (a2b2) and 521 (a1b1) or with a mixture of Δvtc4 a2b2 and Δvtc4 a1b1 strains. The kernels of ears infected with wild-type U. maydis develop into large tumors that are filled with fungal cells differentiating into melanized teliospores (resulting in the tumors' black appearance). Likewise, ears infected with a Δvtc4 a2b2-Δvtc4 a1b1 cross also produced large tumors filled with melanized teliospores. The levels of teliospore production were similar in ears infected with mixtures of wild-type or mutant strains. (B) Teliospores isolated from tumors of plants infected with crosses of wild-type strains 518 (a2b2) and 521 (a1b1) or with a mixture of the Δvtc4 a2b2 and Δvtc4 a1b1 strains. The morphology of teliospores isolated from plants infected with the mutants was indistinguishable from that of teliospores from a wild-type infection. Images were captured by DIC microscopy. Scale bars = 20 μm.

FIG. 6.

Deletion of vtc4 suppresses the multibudded phenotype of a ubc1 mutant. Wild-type, Δvtc4, Δubc1, or Δvtc4 ubc1 cells were grown in MM plus 1% glucose and 1, 7.35, or 250 mM KH₂PO₄ (pH 7). Wild-type U. maydis grows as budding yeast regardless of the extracellular phosphate concentration. The Δvtc4 mutants grow as budding yeast in 1 mM KH₂PO₄, as elongated cells in 7.35 mM, and as hyphal filaments in 250 mM KH₂PO₄. The Δubc1 mutant exhibits a multibudded phenotype, regardless of the extracellular phosphate concentration. The Δvtc4 ubc1 double mutants have the same cellular morphology as the Δvtc4 single mutants. In 1 mM KH₂PO₄, Δvtc4 ubc1 mutant cells grow predominately as budding yeast cells. The Δvtc4 ubc1 cells grown in 7.35 mM KH₂PO₄ have an elongated cellular morphology, and cells grown in 250 mM KH₂PO₄ exhibit a hyphal morphology. Images were captured by DIC microscopy. Scale bars = 20 μm.