The Gbeta-subunit-encoding gene bpp1 controls cyclic-AMP signaling in Ustilago maydis

Abstract

In the phytopathogenic fungus Ustilago maydis, fusion of haploid cells is a prerequisite for infection. This process is controlled by a pheromone-receptor system. The receptors belong to the seven-transmembrane class that are coupled to heterotrimeric G proteins. Of four Galpha subunits in U. maydis, only gpa3 has a function during mating and cyclic AMP (cAMP) signaling. Activation of the cAMP cascade induces pheromone gene expression; however, it does not lead to the induction of conjugation tubes seen after pheromone stimulation. To investigate the possibility that a Gbeta subunit participates in pheromone signaling, we isolated the single beta subunit gene, bpp1, from U. maydis. bpp1 deletion mutants grew filamentously and showed attenuated pheromone gene expression, phenotypes associated with deltagpa3 strains. In addition, a constitutively active allele of gpa3 suppressed the phenotype of the bpp1 deletion strains. We suggest that Bpp1 and Gpa3 are components of the same heterotrimeric G protein acting on adenylyl cyclase. Interestingly, while deltagpa3 strains are impaired in pathogenicity, deltabpp1 mutants are able to induce plant tumors. This could indicate that Gpa3 operates independently of Bpp1 during pathogenic development.

FIG. 1.

Phylogenetic comparison of Bpp1 of U. maydis to various Gβ subunits of fungi and higher eukaryotes. The bootstrap analysis was performed with Clustal W1.8 (49) with the seven-WD40-repeat-containing protein Cpc2 from S. pombe as an outgroup. Accession numbers are CAB11079.1 for Cpc2 of Schizosaccharomyces pombe, BAC01165 for Mgb1 of Magnaporthe grisea, BAB69489 for Fgb1 of Fusarium oxysporum, O14435 for Gb-1 of Cryphonectria parasitica, AAL90861 for Gpb1 of Sporothrix schenckii, AAM53552 for GNB-1 of Neurospora crassa, AAC33436 for SfaD of Aspergillus nidulans, P36408 for GPBA of Dictyostelium discoideum, and Gpb1 of Coprinus cinereus on Contig 1.28 (http://www-genome.wi.mit.edu/annotation/fungi/coprinus_cinereus/); AAD03596 for Gpb1 of Cryptococcus neoformans, AAN33051 for Bpp1 of U. maydis, NP_002065.1 for GNB1 of Homo sapiens, XP_315941 for Beta-1 of Anopheles gambiae, NP_496508 for Gpb-1 of Caenorhabditis elegans, NP_595613 for Git5p of Schizosaccharomyces pombe, and Ste4p of Candida albicans on Contig6-1885 (http://www.ncbi.nlm.nih.gov/genomes/framik.cgi?taxid=5476); and NP_014855 for Ste4p of Saccharomyces cerevisiae.

FIG. 2.

Exogenous cAMP restores budding growth and mating in Δbpp1-2 deletion mutants. (A) Strains as indicated on the left were grown in PD liquid medium without cAMP (left panels) or with 6 mM cAMP (right panels) for 16 h. Both strains are in the a2 b2 background. Bars, 20 μm. (B) Wild-type strain FB2 (a2 b2) and FB2Δbpp1-2 were incubated for 36 h on a PD-charcoal plate in the absence (left panel) or presence (right panel) of 6 mM cAMP. (C) FB2Δbpp1-2 carrying either pNEBUH-bpp1 (left panel) or the empty vector pNEBUH (right panel) was grown in PD liquid medium containing hygromycin. Bars, 20 μm.

FIG. 3.

Relationship between bpp1 and the four different genes encoding Gα subunits. (A) Strains were all in the a2 b2 background and were grown on charcoal-containing CM plates in the absence (upper panel) or presence (lower panel) of 6 mM cAMP for 36 h. Pictures were taken with an Olympus Binocular. (B) Strains (all in a1 b1 background) were grown in liquid CM (upper panel) for 16 h or on charcoal-containing CM plates (lower panel) for 36 h. Bars, 10 μm (upper panels) or 500 μm (lower panels).

FIG. 4.

Pheromone gene expression in Δbpp1-2 strains. We loaded 15 μg of total RNA per lane and subjected it to Northern analysis. The same filter was hybridized in succession with probes for mfa1 and cbx or rRNA as loading controls. (A) Strains and strain mixtures, indicated at the top, were grown on charcoal-containing CM plates for 48 h. (B) The FB1Δbpp1-2 deletion mutant was grown in liquid PD medium without and with cAMP at the concentrations indicated. (C) Epistatic relationship of bpp1 and ubc1. Strains were grown on charcoal-containing CM plates for 48 h. (D) Mating reactions of bpp1 deletion strains on PD-charcoal plates supplemented with 6 mM cAMP. Dikaryon formation can be observed as white fuzzy filaments. The strain combinations indicated were spotted alone or in combination and incubated for 48 h at 28°C.