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, ⌬vtc4 mutants possessed a filamentous cellular morphology, in contrast to the budding, yeast-like growth of the wild-type parent. The ⌬vtc4 mutants also displayed decreased symptom development and reduced proliferation in planta. The interaction with PKA signaling was further investigated by the generation of ⌬vtc4 ubc1 double mutants. Deletion of vtc4 completely suppressed the multiple-budded phenotype of a ⌬ubc1 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.

Defects in Phosphate Acquisition and Storage Influence Virulence of Cryptococcus neoformans

“…Deletion of VTC4 in C. neoformans abolished polyphosphate formation, thus indicating functional as well as sequence similarity to VTC4 in S. cerevisiae and U. maydis (34,36). Polyphosphate is found in the vacuole, acidocalcisomes, cytosol, mitochondria, nucleus, and cell wall in fungi (47,70,71).…”

“…Polyphosphate was assayed as described by Boyce et al (36). Briefly, RNA was extracted with a citrate buffer and bead beating in a bead mill to break open the cells and release RNA and polyphosphate.…”

“…Total RNA (5 g to 10 g) was loaded onto a native DNA polyacrylamide gel, with subsequent electrophoresis in 1ϫ Tris-borate-EDTA (TBE) buffer. RNA and polyphosphate were fixed with acetate, stained with toluidine blue O, and destained in acetate, as described previously (33,36). Gels were scanned, and Adobe Photoshop 7.0 (Adobe Systems Software Ireland Ltd., Dublin, Ireland) was used to visualize polyphosphate and to determine the amount of polyphosphate in relationship to a polyphosphate (10 g) loading control (type 45; SigmaAldrich, St. Louis, MO) and the amount of loaded RNA.…”

“…Cells store inorganic phosphate as linear chains of polyphosphate when phosphate is abundant. It is known that polyphosphate in yeasts and filamentous fungi is synthesized at the vacuolar membrane by a vacuolar transport chaperone complex with Vtc4 as the polyphosphate polymerase (33,34,36). However, little is known about the synthesis and function of polyphosphate in fungal pathogens of humans, and we therefore examined polyphosphate accumulation in our mutants.…”

“…In this context, we previously showed that cAMP/PKA signaling influenced the expression of genes for phosphate uptake and storage in the fungal pathogen of maize, Ustilago maydis (35). One of the genes encoded Vtc4, and deletion of this gene reduced polyphosphate formation, influenced the transition between yeast and filamentous growth, and attenuated virulence (36).…”