Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptome analysis of a Ustilago maydis ust1 deletion mutant

ABSTRACT: Ustilago maydis, the causal agent of corn smut disease, is a dimorphic fungus alternating between a saprobic haploid budding form, and an obligate pathogenic filamentous dikaryon. Maize responds to U. maydis colonization by producing highly modified tumorous structures and it is only within these plant galls that the fungus sporulates giving rise to melanized sexual spores, the teliospores. Previously we identified a regulatory protein from the APSES family of transcription factors, which we named Ust1, whose absence in yeast cells led to filamentous growth and the production of highly pigmented spore-like structures in culture. In this study, we analyzed the transcriptome of a ?ust1 deletion mutant. To identify genes potentially involved in the sporulation program, we carried out microarray analysis comparing a haploid U. maydis WT strain (1/2) and ust1 (14/25) in vitro. Wild-type and ?ust1 strains were grown in potato dextrose broth (PDB) for 48 h at 30oC. To allow effective comparison with other array data generated by U. maydis researchers, cells were then transferred to liquid array medium (6.25% Holiday salt solution, 30mM L-glutamine, 1% glucose, pH7.0, filter sterilized) at 30 C. Total RNA samples from WT 1/2 and ?ust1 mutant strains grown in array medium for 24 h (mutant’s filamentous phase), and 48 h (mutant’s spore-like structure formation phase) were extracted and purified (Sigma Spectrum Plant Total RNA Kit, Cat.No.STRN50). RNA was sent to NimbleGen, where it was reverse transcribed to cDNA and Cy3 labeled. The cDNA samples were hybridized, microarray chips scanned, and raw data normalized with appropriate controls.

ORGANISM(S): Ustilago maydis

SUBMITTER: Scott Gold

PROVIDER: E-GEOD-54659 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:In fungi, sexual compatibility is controlled by mating type loci that prevent self-fertilization. In the plant pathogenic fungus Ustilago maydis, the b mating type locus encodes a pair of unrelated homeodomain proteins, termed bE and bW. After fusion of two compatible, haploid cells, the bE and bW proteins form a heterodimeric complex, but only if they are derived from different, compatible alleles. The active bE/bW complex is required and sufficient to initiate pathogenic development of U. maydis, which is prerequisite for sexual reproduction of the fungus. However, the role of the b heterodimer during later stages of pathogenic development was unclear. To analyze b function during in planta development, we generated a temperature-sensitive bE allele (bEts) encoding a protein with a single amino acid alteration at the border of the homeodomain. This mutation leads to a stop in pathogenic development at the restrictive temperature in planta, while bEts strains show normal development at permissive temperature. At restrictive temperature, hyphae develop enlarged, bulbous cells at their tips that contain multiple nuclei, indicating a severe defect in cell division. DNA array analysis of bEts mutant strains in planta revealed a b-dependent regulation of genes coding for secreted proteins that were shown to influence fungal virulence. Our data demonstrate that in U. maydis the b heterodimer is not only essential to establish the heterodikaryon after mating of two compatible sporidia and to initiate fungal pathogenicity, but also to sustain in planta proliferation and ensure sexual reproduction. Maize plants were infected with a mixture of either FB1 and FB2 (wild-type), or RAb1ts and RAb2ts (temperature-sensitive b heterodimer) and kept at 22Â°C (permissive conditions). Control samples of FB1/FB2 and RAb1ts and RAb2ts were taken 5 days post inoculation at 22 Â°C (1 replicate each), to see if strains expressing the bts heterodimer show wildtype-like biotrophic development. To measure the impact of the b heterodimer on pathogenic development, infected plants were shifted for 9 hours to resrictive conditions (31Â°C) 111 hours post infection. After the temperature shift FB1/FB2 (3 replicates) infections developed normal, whereas RAb1ts/RAb2ts (3 replicates) infections were not able to further proliferate in planta because of a none-functional b heterodimer.

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Transcriptome analysis of a Ustilago maydis ust1 deletion mutant

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